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CELL LINE: A REVIEW

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Published: 06 July 2017

Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies

Kaja C. G. Berg 1 , 2 ,
Peter W. Eide 1 , 2 ,
Ina A. Eilertsen 1 , 2 ,
Bjarne Johannessen 1 , 2 , 3 ,
Jarle Bruun 1 , 2 ,
Stine A. Danielsen 1 , 2 , 3 ,
Merete Bjørnslett 1 , 2 ,
Leonardo A. Meza-Zepeda 3 , 4 ,
Mette Eknæs 1 , 2 ,
Guro E. Lind 1 , 2 ,
Ola Myklebost 3 , 4 , 5 ,
Rolf I. Skotheim 1 , 2 , 3 ,
Anita Sveen 1 , 2 , 3 &
Ragnhild A. Lothe 1 , 2 , 3

Molecular Cancer volume 16 , Article number: 116 ( 2017 ) Cite this article

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Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies.

We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing ( n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis ( n = 297 proteins) by reverse phase protein microarrays.

The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses . We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA- and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFβ signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately ¼ of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene- and protein- expression was found for several oncogenes in individual cell lines, including MYC and ERBB2 .

Conclusions

This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC .

Colorectal cancers (CRC) are molecularly heterogeneous and can be divided into clinically relevant subtypes associated with patient prognosis and treatment response. At the DNA level, this includes the genomic instability phenotypes microsatellite instability (MSI) and chromosomal instability (CIN), as well as the epigenomic CpG island methylator phenotype (CIMP). About 15% of primary CRCs have MSI, while the rest are microsatellite stable (MSS), most of which have the CIN phenotype. MSI tumors have errors in the mismatch repair machinery and display numerous single nucleotide variants (SNVs) and insertions/deletions (indels) [ 1 ]. CIN tumors typically display aneuploidy with structural and/or numerical aberrations, but the underlying cause(s) remains undetermined [ 2 ]. CIMP tumors overlap to a large extent with MSI and are characterized by widespread hypermethylation of CpG islands [ 3 , 4 ].

At the transcriptional level, several classification schemes have identified biologically distinct subtypes of CRCs [ 5 , 6 , 7 ]. The recent identification of four consensus molecular subtypes (CMS) has provided evidence that the expression subtypes have clinical relevance independent of cancer stage [ 8 ]. Although several genomic aberrations associate with individual CMS groups, including MSI and hypermutation in CMS1 and oncogene amplification in CMS2, a potential genomic basis for the expression subtypes remains elusive. Integrative DNA, RNA and protein level analyses promise to improve our understanding of the biological and clinical importance of the evolving molecular classification of CRC.

CMS classification is heavily influenced by the tumor microenvironment, as demonstrated by strong expression of mesenchymal marker genes in the stroma of tumors of the stem-like/mesenchymal subtype CMS4 [ 9 , 10 ]. However, all four CMS subtypes were recently demonstrated to be represented in in vitro model systems (Sveen et al., submitted), and cancer cell lines may therefore be used to identify the cancer cell intrinsic aberrations characteristic of the four CMS groups. Furthermore, genomic studies and drug sensitivity screening have demonstrated that CRC cell lines in general recapitulate the molecular alterations and pharmacogenomics of primary tumors [ 11 , 12 , 13 , 14 , 15 ]. Accordingly, improved molecular characterization of these in vitro model systems may further increase their value as preclinical models of CRC.

Here we present a resource of information for 34 CRC cell lines by multi-level data integration, including targeted deep sequencing, DNA copy numbers, gene expression, microRNA (miRNA) expression and protein expression. We describe consistent gene/pathway regulation across data types and associate this with known CRC subtypes. Each data set and data combination are presented in accessible tables and figures, emphasizing specific alterations of biological or clinical interest for further experimental studies.

Cell lines – Culturing, processing and analyses overview

Thirty-four CRC cell lines purchased from cell line repositories or kindly provided by collaborators (Additional file 1 : Table S1), were subjected to DNA, RNA and protein analyses (Fig. 1 a and b). Cell lines were cultured as previously described [ 12 ] and harvested at approximately 80–90% confluency. Genomic DNA was extracted either by a standard phenol/chloroform procedure or a magnetic beads protocol (Maxwell 16 DNA purification kit, Promega, Madison, WI, U.S.A.). Cell line authenticity was verified by DNA profiling based on 15 short tandem repeat (STR) loci, using the AmpFLSTR Identifiler PCR Amplification Kit (Thermo Fisher, Waltham, MA) and matched to the profiles from supplier (Additional file 1 : Table S1) where available. MSI and CIMP status was determined according to previously described procedures [ 12 ]. For CL-40 MSI status was additionally assessed using the MSI Analysis System, version 1.2 (Promega, Fitchburg, WI, USA). Total RNA was extracted using the Qiagen AllPrep DNA/RNA/miRNA Universal kit (Qiagen, Hilden, Germany) and quality controlled by the Agilent RNA 6000 nano kit for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, U.S.A.). All RIN values were above 9. Protein lysates were produced from cell pellets at the MD Anderson Cancer Centre RPPA Core Facility.

Overview of the 34 CRC cell lines analyzed and key findings. a The cell lines are grouped according to the gene expression-based CMSs (except Colo320, which has a neuroendocrine origin), and MSI, POLE and CIMP status are indicated. In general, the morphologic appearance of cell lines in CMS1 and CMS4 (for example LoVo and RKO) was mesenchymal, whereas cell lines in CMS2 and CMS3 (for example IS3 and WiDr) appeared more epithelial-like. b The cell lines were analyzed on the DNA, RNA and protein levels as indicated ( blue background ). Bioinformatic analyses ( grey ) were performed both on individual data levels and by integration of two or more data levels. Key findings ( white ) and references to figures and tables with detailed results are given ( green ). CIMP: CpG island methylator phenotype, CMS: consensus molecular subtypes, CNA: copy number aberrations, MSI/MSS: microsatellite instable/stable, OG: oncogene, TF: transcription factor, TS: tumor suppressor, SNV: single nucleotide variant

For all cell lines, DNA copy number, mRNA, miRNA and protein expression profiles were generated. Targeted DNA sequencing was performed for 27 cancer cell lines in addition to Sanger sequencing of selected genes in 31 cell lines. For frequency counts and statistical tests we excluded the neuroendocrine Colo320 and kept only one cell line derived from the same patient, thus excluding IS3, SW620, DLD-1 and WiDr.

Targeted deep sequencing

Sequencing libraries for the “kinome” and selected cancer-relevant genes (totally n = 612 genes; Additional file 1 : Table S2) was generated using the Agilent SureSelect Human Kinome V1 kit (Agilent), and 2 × 101 basepair paired-end sequencing was performed on the Illumina HiSeq 2500 system (Illumina, San Diego, CA, U.S.A.) at the Oslo University Hospital Genomics Core Facility to an average sequencing depth of 161X (range 105-289X). Sequencing reads were aligned to the reference genome GRCh37 (hg19) with the Burrows-Wheeler Aligner (v0.6.21) [ 16 ], converted from sequence alignment map (SAM) files to the binary alignment map (BAM) format by Picard, version 1.61 [ 17 ], and sorted and indexed using SAMtools (v0.1.18) [ 18 ]. Duplicate reads were removed by Picard, and the Genome Analysis Toolkit (GATK, v2.7–4) [ 19 , 20 ] was used for local realignment around indels. Variant calling of both SNVs and indels was done using the HaplotypeCaller tool from GATK, and candidate variants were annotated using ANNOVAR (build 2013–02-21) [ 21 ]. Only variants with minimum 10 alternative reads were included in further analysis. As sequencing analyses on cell lines do not enable filtering of germ line variants, candidate variants present in dbSNP version 138 [ 22 ] and not marked as clinically associated in this version of dbSNP, were discarded, and the final cell line “mutations” are referred to as variants throughout the paper. The following variants were defined as non-synonymous: non-synonymous SNVs, stopgain SNVs, stoploss SNVs and frameshift indels.

Sanger sequencing was performed for the whole coding sequences of PTEN and TP53 and for mutation hotspots in KRAS codons G12, G13, Q61, K117 and A146, BRAF V600 and PIK3CA E542, E545, E546, H1025 and H1047 for seven of the cell lines. The mutation statuses for most of the codons above for the remaining 24 cell lines are described previously [ 12 ], except for KRAS codons K117, A146 and PIK3CA codon and H1025, which are included in the current work. Colo205, HCC2998 and KM12 were not assessed by Sanger sequencing.

High resolution DNA copy number profiles

DNA copy number data was generated using Affymetrix Genome-Wide Human SNP 6.0 microarrays (Affymetrix Inc., Santa Clara, CA). One μg of DNA in low-EDTA TE-buffer was prepared according to the Affymetrix SNP 6.0 Cytogenetics Copy Number Assay User Guide and hybridized to Affymetrix Genome-Wide SNP 6.0 microarrays according to the Affymetrix Genome-Wide Human SNP Nsp/Sty User Guide. Resulting raw data were within recommended QC thresholds (CQC > 0.4; MAPD < 0.35). Signal extraction and pre-processing of raw data was performed as previously described [ 23 ], using the PennCNV protocol modified for Affymetrix genotyping arrays with Affymetrix Power Tools version 1.15.0 [ 24 , 25 ] with HapMap samples as reference [ 26 ]. Single-sample segmentation of normalized and GC corrected data was done with the R package copynumber (version 1.14.0) [ 27 ]. The user defined penalty parameter was set to 100. PCF value thresholds were set to ≥0.15 (gain) and ≤ −0.15 (loss). To enable comparison of samples with different breakpoints, the smallest regions of overlap (SROs) were determined. Each SRO originated from a true larger segment and the copy number value of the originating segment was kept. Copy number estimates per gene were retrieved by mapping chromosomal segments from each sample to the R implemented transcript database TxDb.Hsapiens.UCSC.hg19.knownGene (v3.2.2), utilizing the findOverlaps function from the GenomicRanges R package (v1.22.4).

The percentage of the genome affected by copy number aberrations (CNAs) was defined as the percentage of bases with aberrant copy number out of the total number of bases with a copy number value available.

To detect potential CNA targets, The GISTIC algorithm v2.0.22 [ 28 ] was run with default parameters with the following exceptions: the threshold for broad events was set to 70% of the chromosome arm length; the maximum number of segments in a sample was set to 2000; the confidence level was set to 99%; parameters for gene-level and broad-level analysis was set ON.

Gene expression analysis

Microarray gene expression analyses were performed using Affymetrix HTA 2.0 Transcriptome Arrays (Affymetrix Inc., Santa Clara, CA, U.S.A.), according to the manufacturer’s instructions. The data was normalized and summarized at the gene level using the Guanine Cytosine Count Correction and Signal Space Transformation algorithms with Robust Multi-array Average (SST-RMA) implemented in the Affymetrix Expression Console Software (v1.4.1, HTA-2_0.r3 library files). The HTA-2_0.na35.2.hg19.transcript.csv annotation file identified 67,528 annotated genes (transcript clusters). The data was filtered to exclude non-coding RNA probes, and genes annotated by multiple probesets were filtered to retain one probeset per gene by prioritizing annotation databases: RefSeq, ENSEMBL, other databases. The filtered dataset contained data for 18,740 probesets.

Principal component analysis (PCA) was performed including only the 1000 genes with the largest cross-sample variation. PC1 had a bimodal density distribution and samples with PC1 score larger than the between-peaks minima were defined as “high”. Gene set tests were performed using camera [ 29 , 30 ]. Single sample Gene Set Enrichment Analysis (ssGSEA) was performed using GSVA [ 31 ]. Seventy gene sets were assembled to enrich for pathways likely to be informative on CRC biology based on Guinney et al. [ 8 ] (Additional file 1 : Table S3). Differential expression analysis was performed using the R package limma [ 30 ].

The cell lines have been classified according to CMS based on the nearest predicted subtype, using an adapted classifier independent of gene expression signaling from the tumor microenvironment (Sveen et al., submitted).

The gene expression data has been submitted to the NCBI’s Gene Expression Omnibus with accession number GSE97023.

Small RNA sequencing

Small RNA sequencing libraries were prepared using a recently published low-bias protocol [ 32 ] and resulting libraries subjected to sequencing on an Illumina HiSeq 2500 (rapid mode). The 50 bp single-end reads were de-multiplexed and converted to FASTQ files by Casava (v1.8.2). Reads were adapter trimmed, quality filtered and collapsed by FASTX Toolkit (v0.0.14). We discarded reads that met any of the following criteria; less than 7 bases matching adapter sequence, shorter than 18 bases after adapter clipping, and/or phred score below 27 for more than 8% of the bases. The eight randomized N-bases were removed prior to alignment. Processed reads were aligned against a custom reference of miRBase hairpins (v21) using bowtie (v1.1.1), allowing no mismatches. The reads were summarized over each mature miRNA, requiring at least 18 nt overlap using R packages GenomicRanges, rtracklayer and ShortRead. Differential expression analysis of miRNA data was performed with R package limma using voom with cyclic loess normalization [ 30 , 33 ].

High-throughput protein expression analysis

Reverse Phase Protein lysate Array (RPPA) analysis with 297 antibodies targeting 235/62 proteins/phospho-proteins (Additional file 1 : Table S4) was performed at MD Anderson Cancer Centre RPPA Core Facility, including pre-processing of the protein data. Median centered normalized log 2 values describing the relative protein abundance in each sample were used for downstream analyses. Differential expression analysis of RPPA data was performed with R package limma [ 30 ].

Integration of DNA copy number and gene expression data

We explored the influence of in-cis copy number aberrations on gene expression by testing for differences in gene expression among CNA groups. For this analysis, a stricter PCF value threshold of ≥0.3 or ≤ − 0.3 was used to define gain and loss. Genes spanning several segments (differing in PCF value along the gene) were handled as follows: Genes consistently gained or lost (different PCF value but belonging to the same category) were assigned to the correct category accordingly. Genes that differed in copy number category (e.g. loss in one part, gain or neutral in remaining part) was assigned the median PCF value. Only genes represented in all samples were included. The mRNA expression was defined to be associated with copy number in cis if Wilcoxon testing determined (i) the mRNA expression to be significantly different in samples with gain versus samples with neutral copy number or loss, or (ii) the mRNA expression was significantly different in samples with loss versus samples with neutral copy number or gain. We corrected for multiple testing by false discovery rate (FDR) using the p.adjust function in the R stat package.

To limit false positives, genes within the lower quartile of mean gene expression were excluded and only genes with IQR > 0.7 were retained, n = 5120 genes for in cis analyses. Only unique MSS cell lines ( n = 18) and genes with aberrant copy number > 2 cell lines were investigated (gain: 1148 genes; loss: 1047 genes). GO enrichment analysis of significant in cis genes was done with the PANTHER overrepresentation test with the GO consortium online tool [ 34 ]. Significant genes from in cis analyses were investigated for overlaps with the MSigDB version 5.2 [ 35 , 36 ].

Associations between CNA and gene expression were additionally assessed by gene-wise Spearman correlations of copy number- and expression values across samples, and genes with correlations above 0.7 were considered to show an association.

To identify potential CNA drivers, we looked for outliers in CNA estimates corresponding to high or low in-cis expression in unique CIN cell lines. We applied a cutoff of 4 times gain/loss threshold (0.15/−0.15) to nominate potentially high amplitude CNAs, and gene expression values outside 3 times the standard deviation from sample mean expression across all genes were considered outliers and hence interesting. We looked for concurrent CNAs and gene expression events by retrieving genes for which the minimum/maximum CNA value and gene expression value belonged to the same cell line.

A panel of 34 CRC cell lines was analyzed at the DNA, RNA, and protein level (Fig. 1a ; Additional file 1 : Table S1). Results are presented in figures and tables for each individual data level and integration analysis, as summarized in Fig. 1b . The panel comprised 11 MSI and 22 MSS cell lines, in addition to the MSS POLE mutated HCC2998 [ 37 ]. The cell lines have previously been shown to recapitulate the biological properties of the four CMSs (Sveen et al., submitted). Out of the 34 cell lines, 8 were classified as CMS1-“immune”, 9 as CMS2-“canonical”, 6 as CMS3-“metabolic”, and 10 as CMS4-“mesenchymal”. Colo320 is derived from a neuroendocrine tumor and has a distinct gene expression profile [ 38 ].

DNA sequence aberrations reflect hypermutator phenotypes

Cell lines with a hypermutator phenotype associated with MSI or POLE mutation had a median of 126 (range 98–327) non-synonymous variants (SNVs or indels) in the 612 sequenced genes, significantly more than the 18 (range 4–26) found in MSS cell lines ( p = 9∙10 −5 , Wilcoxon rank-sum test). This corresponds to 82 (range 63–212) and 12 (range 3–17) non-synonymous variants per million coding basepairs sequenced, respectively. For reference, The Cancer Genome Atlas reported approximately 1–300 somatic mutations per million basepairs for primary CRCs [ 39 ]. MSI cell lines had a high proportion of C > T variants, especially in an NpCpG sequence context, consistent with a mismatch repair deficiency mutation signature commonly found in MSI cancers (Signature 6; Fig. 2a ) [ 40 ]. The MSI cell lines DLD-1 and HCT15 (derived from the same patient) had the highest variant loads, with 592 and 442 SNVs respectively (Additional file 2 : Fig. S1a). In addition to the large proportion of MSI-associated C > T variants, these cell lines had a larger contribution of C > A variants in a CpCpT sequence context compared to other MSIs, recently reported to be caused by a POLD1 R689W mutation (Fig. 2a ) [ 41 ]. Consistently, DLD-1 and HCT15 also had a substantially lower number of indels relative to SNVs than other MSI cell lines (Additional file 2 : Fig. S1a). The MSS cell line HCC2998, which has a POLE P286R substitution [ 37 ], had the third highest variant load with 281 non-synonymous variants. This cell line had few indels and a high proportion of C > A variants in a TpCpT context, C > T variants in a TpCpG and T > G variants in a TpTpT context, which are associated with the POLE hypermutator phenotype and mutation Signature 10 [ 40 ].

DNA aberrations reflect the type of genomic instability. a We investigated the frequencies ( vertical axes ) of SNVs in each of six categories (indicated in the top panels) grouped according to sequence motif (flanking nucleotides are indicated on the horizontal axes). MSI cell lines ( n = 8, excluding DLD1 and HCT15) and the POLE mutated cell line HCC2998 displayed different mutation signatures associated with the respective types of hypermutation. The MSI cell lines DLD-1 and HCT15 had a distinct mutation signature with a combination of deficient mismatch repair and POLD1 mutation. b Overview of detected SNVs/indels in 37 genes included in the Cosmic Cancer Gene Census and that were mutated in at least four MSI cell lines or one MSS cell line among the 27 cell lines analyzed by targeted deep sequencing. Most genes showed clear mutation frequency differences between MSS and MSI/ POLE mutated cell lines. c There was an inverse relationship between the CNA load (horizontal axis; percent of basepairs with aberrant copy number) and the SNV/indel load ( vertical axis ) in the cell lines, reflecting their molecular subtype, as indicated. The neuroendocrine cell line Colo320 ( green circle ) grouped along with the MSS cell lines, and had few SNVs/indels and a moderate number of CNAs, including gain of 8q and 13q. d MSI/ POLE mutated cell lines had a lower frequency of CNAs ( vertical axis ) along the genome than e MSS cell lines. In each plot, chromosomes are indicated on the horizontal axes and separated by vertical lines (whole and dashed lines for chromosomes and chromosome arms, respectively). Frequent aberrations are highlighted, including gains on 7p, 7q, 8q, 12p, 13q, 20q and losses on 4p, 4q, 17p, 18q and 22q, which are chromosome arms known to be frequently affected by CNAs in primary CRCs. CNA: copy number aberration, MSI/MSS: microsatellite instable/stable, POLE: POLE mutated, SNV: single nucleotide variant

The genes that were most frequently affected by SNVs/indels and also listed in the COSMIC Cancer Gene Census are summarized in Fig. 2b . Selected variants in CRC critical genes, analyzed by Sanger and/or targeted sequencing, are presented in Table 1 . None of the detected common variants were restricted to one CMS group, and variant frequencies rather reflected the MSI status of the cell lines. A complete list of the detected exonic non-synonymous SNVs and indels is found in Additional file 1 : Table S5.

DNA copy number aberrations reflect the CIN phenotype

We confirmed an inverse relationship between the number of SNVs/indels and DNA copy number aberrations (CNAs, % genome affected), reflecting the type of genomic instability (Spearman’s rho = −0.74, p = 1∙10 −5 ; Fig. 2c ). MSI/ POLE mutated cell lines had significantly less CNAs (range 0–14%, median 9%) compared to MSS cell lines (range 12–69%, median 40%; Wilcoxon rank-sum test, p < 2.2∙10 −16 , Additional file 2 : Figure S1b; Additional file 1 : Table S1). CL-40, which is previously reported to have MSI [ 14 ], was here found to be MSS, but the number of CNAs was low and the cell line may thus represent a non-CIN non-MSI phenotype (12% genome affected by CNAs). CMS1 cell lines had fewer CNAs (range 0–45%, median 10%) compared to CMS2/3/4 (range 7–69%, median 32%; Wilcoxon rank-sum test, p = 0.01), reflecting the high prevalence of MSI in the CMS1 subtype, and CMS2 cell lines had more CNAs compared to CMS1/3/4, although not statistically significant (CMS2 range 27–59, median 33%; CMS1/3/4 range 0–69, median 13%; p = 0.06).

Although cell lines with MSI or POLE mutation ( n = 11) harbored few DNA copy-number aberrations, two broad gains and four focal losses were observed with frequencies higher than 40% (Fig. 2d ). In contrast, MSS cell lines ( n = 18) had 24 separate regions affected in more than 40% of the cell lines (Fig. 2e ). CNAs detected in MSI cell lines were not exclusive for this subtype, although the focal losses were less frequent in MSS cell lines.

Potential target genes of CNAs were identified in 7 and 23 focal areas of gain and loss respectively (GISTIC analysis, q-value < 0.25; Additional file 1 : Table S6), including KLF5 (gain 13q), GPHN (loss 14q) and SMAD4 (loss 18q), as well as genes located in known fragile genomic areas, like FHIT (3p), WWOX (16q) and MACROD2 (20p).

No copy number changes were restricted to one CMS group (MSS only; Additional file 2 : Figure S1c). Some CNAs were more recurrent in undifferentiated MSS cell lines ( n = 6, mainly CMS1 and CMS4 cell lines) compared to the colon-like MSS cell lines ( n = 12, mainly CMS2 and CMS3) and vice versa. This included higher frequency of chromosomes 8 and 13 gain and loss of focal regions on 3p, 4q, 14q, 17p, 20p and 22q in colon-like cell lines and gain of 5q and 22q in undifferentiated cell lines (Additional file 2 : Figure S1d). A genome wide overview of gene copy number status for all cell lines is presented (Additional file 1 : Table S7).

mRNA, miRNA and protein expression profiles are distinct between undifferentiated and “colon-like” cell lines

Unsupervised PCA of mRNA expression data showed that the cell lines formed two distinct clusters, as highlighted by the bimodal density distribution of samples along the first principal component (PC1, Fig. 3a ). A similar pattern was apparent also in the miRNA and protein expression datasets (Additional file 3 : Figure S2a). To explore the biological basis for this separation, we correlated PC1 from mRNA expression data to single-sample gene set enrichment analysis (ssGSEA) scores for 70 pre-selected CMS and CRC relevant gene sets (Additional file 1 : Table S3). The top hit was a gastro-intestinal tissue enhanced gene set, derived from The Human Protein Atlas [ 42 ], with the ssGSEA score explaining more than 90% of the variance along PC1 ( r 2 = 0.92, p < 2∙10 −16 , Pearson’s correlation, Fig. 3b ). We used the PC1 density to classify the cell lines with low PC1/high gastro-intestinal ssGSEA score as colon-like and the remaining as undifferentiated (18 and 15 cell lines, respectively) . This grouping was significantly associated with the CMS groups (CMS2/3 versus CMS1/4), but less so with MSI-status ( p = 2∙10 −6 and p = 0.06, respectively, Fisher’s exact test). The finding was corroborated by morphological appearances; for example the undifferentiated cell lines LoVo and RKO appeared more mesenchymal, while colon-like IS3 and WiDr formed large epithelial-like sheets in culture (Fig. 1a ). To further characterize the differences between colon-like and undifferentiated cell lines, we performed gene set analysis [ 29 ]. Out of 70 gene sets, 17 showed a significant relative difference (FDR corrected p < 0.05, Fig. 3c and Additional file 1 : Table S3). In addition to the gastro-intestinal markers, colon-like cell lines were characterized by relative upregulation of genes positively regulated by the HNF4A and CDX2 transcription factors and genes repressed by HNF1A and WNT signaling (Fig. 3c ). Conversely, undifferentiated cell lines had higher epithelial-to-mesenchymal transition (EMT) signature score and increased expression of TGFβ induced genes (Fig. 3c ).

Gene expression based classification of CRC cell lines revealed a separation between colon-like and undifferentiated cell lines associated with the consensus molecular subtypes (CMS). a PCA of cell line mRNA expression data (plotted as sample-wise PC1 versus PC2) showed that the cell lines had a bimodal density distribution along PC1 ( bottom plot ), indicating two distinct subgroups largely separating CMS2/3 from CMS1/4. Each point represents one cell line, and is colored according to the CMS class and with point type indicating MSI-status. Dashed vertical line ( red ) indicates the least frequent value between the two density modes of PC1, and was used as a threshold to separate the cell lines into the two subgroups. b PC1 ( horizontal axis ) was strongly correlated with the sample-wise enrichment score for a set of gastro-intestinal tissue enhanced genes ( vertical axis ), and cell lines with high enrichment scores, left of the red dashed line, were termed “colon-like” and the remaining “undifferentiated”. c Gene set enrichment analyses comparing colon-like and undifferentiated cell lines showed that colon-like cell lines had higher expression of genes upregulated by HNF4A and lower expression of genes related to colorectal cancer stemness. Undifferentiated cell lines had higher expression of genes related to epithelial to mesenchymal transition and genes upregulated by TGFβ. The plot includes the top 15 gene sets tested (ranked by p -value) and the -log 10 p -value is plotted on the horizontal axis. d Top 5 differentially expressed transcription factors and kinases (mRNA level), miRNAs and proteins between colon-like and undifferentiated cell lines. mRNAs and miRNAs are ranked by p -value while proteins are ranked by absolute log 2 fold-change. The log 2 fold-changes (log 2 FC) between the sample groups are indicated. e Classification of the individual cell lines according to the colon-like and undifferentiated subgroups. CRC: colorectal cancer, CMS: consensus molecular subtypes, log 2 FC: log 2 fold-change, MSI/MSS: microsatellite instable/stable, PCA: principal component analysis

To pinpoint important factors maintaining the distinction between colon-like and undifferentiated cell lines, we performed differential mRNA, miRNA and protein expression analysis (Additional file 3 : Figure S2b, Additional file 1 : Tables S8, S9, S10). At the mRNA level, CEACAM5, which encodes a carcinoembryonic antigen (CEA) protein used as a blood-based biomarker for monitoring CRC patients, was more than 100-fold higher in colon-like cell lines. In undifferentiated cell lines, TGFB1 and TGFB2 were 3- and 7-fold higher, respectively. The five transcription factors with the most significant upregulation in colon-like cell lines were MYB, MECOM, ETS2, HNF4A and CDX1 (Fig. 3d ), consistent with high expression of these genes in human gastro-intestinal tissues [ 42 ]. The most significantly upregulated transcription factors in undifferentiated cell lines were SIX4 , ZNF286A , MSX1 , ZNF286B and MLLT10 .

Differential miRNA expression analysis showed upregulation of the miRNAs encoded in the mir-194 ~ 192 and mir-200b ~ 429 clusters in colon-like compared to undifferentiated cell lines. MiRNAs in the mir-194 ~ 192 cluster are highly specific to colonic tissue [ 43 ] while miR-200 is critical in establishing and maintaining epithelial cell identity [ 44 ], corroborating the mRNA-based subgroup designations. Among proteins analyzed, AXL, CAV1, ANXA1, phosphorylated RPS6 and L1CAM (CD171) were highly upregulated in undifferentiated cell lines (Fig. 3d , Additional file 3 : Figure S2b). For colon-like samples, MUC1, UGT1A, RAB25, SYK and β-catenin (CTNNB1) had the largest fold-change when compared to undifferentiated cell lines, but also E-cadherin (CDH1) and EGFR were significantly upregulated.

Summarized, CRC cell lines form two major biologically distinct expression subgroups at the mRNA, miRNA and protein level, which are distinguished by the expression of gastro-intestinal and epithelial differentiation markers.

Integrated analysis identifies in vitro models for studies of targetable genes

To detect genes and pathways repeatedly affected by different aberrations in individual cell lines, we integrated data from different genomic levels, focusing on central CRC pathways and transcription factors.

Concurrent CNAs and SNVs/indels in cancer critical genes

In some cell lines, a simultaneous SNV/indel and CNA in the same gene was observed, including gains and SNVs in the oncogenes KRAS ( n = 6 cell lines) and EGFR ( n = 2). A complete overview of SNVs/indels, CNAs and the combination of these events in individual cell lines is shown in Fig. 4 ( n = 83 genes in the Cancer Gene Census represented in the targeted sequencing data).

CNAs and SNVs/indels in cancer-critical genes. Among genes in the Cancer Gene Census ( n = 83 genes included in the targeted sequencing panel, ranked vertically in alphabetical order), simultaneous mutations and CNAs in individual cell lines (grouped horizontally according to genomic phenotypes as indicated) were detected in CRC relevant oncogenes, including KRAS and EGFR , and tumor suppressor genes, including TP53 and APC . The cell line Colo320, which has a neuroendocrine origin, is marked by an asterisk. CNA: copy number aberration, CRC: colorectal cancer, MSI/MSS: microsatellite instable/stable, POLE: POLE mutated, SNV: single nucleotide variant

mRNA expression of oncogenes and transcription factors is associated with DNA copy number in cis

A total of 298 (26%) out of 1148 genes with copy number gain had a significant in cis association between copy number state and gene expression (analyzed by Wilcoxon rank-sum test) (Additional file 1 : Table S11). Out of the 298 genes, 215 (72%) had strong correlations between copy number estimates and gene expression (Spearman correlation >0.7), 10 of which were found in the COSMIC Cancer Gene Census and 25 defined as transcription factors in the Molecular Signatures Database (MSigDB; highlighted in Additional file 1 : Table S11). Similarly, 229 out of 1047 (22%) genes with copy number loss had an in cis association with gene expression (Additional file 1 : Table S11). Out of the 229, 174 (76%) showed strong correlations between copy number estimates and gene expression (Spearman correlation >0.7), and 8 were found in the COSMIC Cancer Gene Census and 15 defined as transcription factors in MSigDB (highlighted in Additional file 1 : Table S11). For gained in cis genes, the largest fold enrichment from gene ontology analysis was found for genes involved in nucleic acid metabolic process and cellular protein metabolic process (>1.5-fold enrichment). Biological processes enriched among lost in cis genes were mitotic cell cycle process, protein transport and intracellular transport (>2-fold enrichment; Additional file 1 : Table S12).

Among the genes with significant in cis copy number and gene expression regulation, 56 were differentially expressed between colon-like and undifferentiated cell lines, including the transcription factors ELF1 and KLF5 and the lysosomal marker LAMP1 (higher expressed in colon-like cell lines; FDR corrected p < 0.05; Additional file 1 : Table S11).

Gene amplification and outlier expression

Genes with high-level copy number amplification and concurrent outlier gene expression may represent potential driver genes and drug targets. We identified 280 such genes across 18 unique MSS cell lines (Additional file 1 : Table S13). Of these, 22 genes were classified as transcription factors in MSigDB and 15 genes were found in the COSMIC Cancer Gene Census, including ERBB2 (Colo678), MYC (SW480), PPFIBP1 (IS1), and RAD21 (HT29) (Additional file 4 : Figure S3a). The cell line V9P had high-level amplification with concurrent high expression of 68 genes, 29 of which were located on 22q, including SMARCB1, BCR and MIF (Additional file 1 : Table S13). Protein expression data were available for ten genes, confirming high expression also at the protein level of the majority, including ERBB2, CCNE1 and MYC, suggesting that these copy number events are functionally important (Additional file 4 : Figure S3b).

Consistent expression regulation at the gene and protein level

To assess the correspondence between mRNA and protein-level expression (RPPA data) we calculated the Pearson’s correlations for each gene-protein pair among the cell lines (Fig. 5a ). Excluding gene-protein pairs for which there was little variation among cell lines (lowest quartile in either dataset), the median correlation for all investigated pairs was 0.59 (IQR 0.26–0.78). AXL, CAV1, CDH1 (E-cadherin), EGFR and L1CAM had very strong correspondence, with correlation coefficients above 0.9. Similarly, mRNAs that were differentially expressed between colon-like and undifferentiated cell lines were generally also differentially expressed at the protein level (Fig. 5b ).

mRNA and protein expression levels are highly concordant among cell lines. a The density distribution ( horizontal axis ) of cross-cell line Pearson’s correlations ( vertical axis ) for expression of matched genes (microarray data) and proteins (Reverse Phase Protein Array data) ( n = 194) shows an overall strong correlation. The horizontal line indicates the median correlation coefficient for all gene-protein pairs. b Differential expression analyses between colon-like and undifferentiated cell lines showed strong correspondence at the mRNA and protein level (plotted as the log 2 fold-changes between the two groups of cell lines for matched protein on the vertical axis versus mRNA on the horizontal axis). The plot includes gene-protein pairs with adjusted p -value <0.1 from differential expression analysis in either mRNA or protein data. Gene-protein pairs with absolute log 2 fold-change >0.5 (mRNA) between colon-like and undifferentiated cell lines are indicated by names and the rest by circles. Pearson correlation analysis (r 2 ) indicated that 43% of the variance in the log 2 fold-change at the protein level could be explained by mRNA-level log 2 fold-change

Multi-level data integration emphasizes molecular differences among cell lines relevant for functional studies

To facilitate selection of cell lines as appropriate research models, we performed gene expression enrichment analysis of eight gene sets representing important pathways or processes in CRC, including ERK/MAPK, PI3K/AKT, EGFR, TGFß and WNT signaling, in addition to signatures of epithelial to mesenchymal transition, citric acid cycle activation and the gastro-intestinal signature (Fig. 6a ; Additional file 1 : Table S14). The resulting heatmap indicates favorable systems for studying particular aspects. For example, SW1463 and CL40 are well-differentiated with low EMT-signature compared to CaCo2 and LoVo. Similarly, Colo205 and SW1116 have relatively low intrinsic TGFß activation in contrast to SW48 and CL-11. Finally, we assembled a list of outlier characteristics from the other data levels (Fig. 6b ). Striking examples include high expression of the immune-suppressive protein PD-L1 in RKO, as well as mutation and downregulation of PTEN in KM12 and Co115, with concomitant hyper-phosphorylation of the AKT protein at residue T308.

Characteristics of individual cell lines at multiple molecular levels. The cell lines are ranked alphabetically within the colon-like ( n = 18; top) and undifferentiated ( n = 15; bottom) subgroups. The neuroendocrine Colo320 is found below the undifferentiated cell lines (marked by a dark grey box). a The heatmap shows standardized single sample gene set expression enrichment scores for the eight selected pathways indicated at the bottom (indicates how many standard deviations the score is above or below the mean). Red indicates relative upregulation and blue indicates relative downregulation among cell lines. b The table indicates selected molecular events characteristic of each cell line. Amp: DNA amplification, mut: “mutation” (single nucleotide variant or insertion/deletion), m: mRNA level, p: protein level, wt: wild type

CRC cell lines have previously been shown to recapitulate the mutational and transcriptional heterogeneity of primary tumors [ 7 , 12 , 14 , 45 ]. Here we report an expanded overview of DNA, RNA and protein level characteristics of 34 CRC cell lines, analyzed in relation to genomic instability phenotypes and gene expression subgroups.

Consistent with known characteristics of the MSI and CIN phenotypes, we observed inverse levels of SNVs/indels and CNAs. All MSI cell lines had a mismatch repair deficiency-associated mutation signature, however, DLD-1 and HCT15 additionally had a high contribution from C > A variants in CpCpT trinucleotides and a low indel burden, a phenotype recently found to be the caused by the combination of MSI and a POLD1 R689W mutation [ 41 ]. Although mutation analysis was restricted to a panel of 612 genes, the high mutation load in MSI/ POLE mutated cell lines allowed for detection of expected mutation signatures. In the cell lines with a lower mutation load, broader sequencing coverage (whole exome or genome sequencing) would be more appropriate for accurate analyses of mutation processes. Unexpectedly, we find no evidence of MSI in CL-40, which has previously been reported as an MSI cell line [ 14 ]. This cell line also had a low number of CNAs, indicating that it may represent a non-MSI, non-CIN genomic phenotype.

Two distinct subgroups of CRC cell lines were evident at the mRNA, miRNA and protein expression levels. Based on gene set associations we termed these groups colon-like and undifferentiated . Colon-like cell lines were either CMS2 or CMS3, expressed higher levels of gastro-intestinal marker genes, including key transcription factors such as HNF4A and MYB . HNF4A has been nominated as a candidate driver for the 20q13.12 focal amplification peak suggesting a possible causal relationship between overexpression and expression subtype [ 39 , 46 ]. Colon-like samples had in addition higher expression of mir-194 and mir-192, both highly enriched in colonic mucosa compared to other human tissues [ 43 ], independently supporting differentiation as a key distinction between the two cell line subgroups. Further, the miR-200 family, which represses the epithelial to mesenchymal transition program [ 44 ] was among the most abundant and most significantly upregulated miRNAs in the colon-like samples. Mir-200 promoter hypermethylation with concomitant downregulation was recently suggested to be a candidate marker for CMS4 tumors [ 47 ].

All CMS4 and most CMS1 models were classified as undifferentiated, consistent with primary tumors where CMS1 and CMS4 display a more stromal and undifferentiated signature [ 8 ]. As a group, undifferentiated cell lines showed relative upregulation of epithelial to mesenchymal transition signature and increased expression of TGFβ induced genes including TGFβ1/2 cytokines. Recently it was shown that TGFβ signaling in cancer associated fibroblasts (CAFs) promotes tumor initiating capacity of CRC cells, and that CRC organoids with high TGFβ expression has a high metastatic potential [ 10 ]. As such, CMS1/CMS4 cancer cells may induce pro-metastatic behavior of CAFs through TGFβ1/2 paracrine signaling, illustrating how cancer cell-intrinsic expression may modulate the tumor microenvironment.

The notion that poorly differentiated tumors have inferior prognosis is not new [ 48 , 49 , 50 ] and the undifferentiated CMS4 is of particular clinical interest due to its association with poor prognosis. As such, the traits described here may be useful for further detailed studies of the biological background of this subtype. Also clinically relevant, undifferentiated cell lines expressed lower levels of CEA than colon-like cell lines, an observation which suggests that this biomarker may be less valuable in monitoring patients with CMS1 and CMS4 cancers.

Recurrently amplified chromosomal regions may harbor oncogenes that become overexpressed from the increase in gene-dosage [ 51 ]. About ¼ of genes had a good correlation between copy number state and gene expression level, some of which were also differentially expressed between colon-like and undifferentiated subtypes, such as the transcription factor KLF5 (higher expressed in colon-like cell lines). Additionally, we identified high level amplifications with concurrent high gene expression in individual cell lines, including ERBB2 in Colo678, also corroborated by high protein expression. The use of HER2-inhibitors together with the kinase inhibitor lapatinib was recently described as a treatment option in HER2 amplified, KRAS wild-type metastatic CRC in a phase 2 trial [ 52 ]. We also observe that Colo678 have high ADAM10 expression, suggested to be involved in acquired resistance to HER2-inhibition in breast cancer models. [ 53 ], and Colo678 may be used as a model system to elucidate resistance mechanisms for HER2 inhibition in CRC. V9P has few SNVs/indels, and drivers in this cell line are not well-explored. We found V9P to have concurrent amplification and high gene expression for more than 60 genes, including CCNE1 (cyclin E1), which also had concurrent high protein expression levels. V9P represents a model for overexpression of cyclin E1, commonly observed in many cancers and which has been linked to chromosome instability [ 54 , 55 ].

By integration of DNA, RNA and protein data, we show that CRC cell lines represent consistent molecular subgroups defined by genomic instability phenotypes at the DNA level (sequence aberrations and CNAs) and differentiation at the expression level (mRNA, miRNA and protein). The data are made available per cell line in summary illustrations and detailed tables, and is a resource to select relevant models for further studies of cancer-cell intrinsic differences among CMS groups, functional biological mechanisms of CRC as well as pharmacogenomics.

Abbreviations

cancer associated fibroblasts

carcinoembryonic antigen

CpG island methylator phenotype

chromosomal instability

consensus molecular subtype

copy number aberration

colorectal cancer

epithelial-to-mesenchymal transition

false discovery rate

insertion or deletion

microsatellite instability

molecular signatures database

microsatellite stability

principal component analysis

robust multi-array average

reverse phase protein lysate array

single nucleotide variant

smallest region of overlap

single sample gene set enrichment analysis

signal space transformation

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Acknowledgements

We thank the MD Anderson Cancer Centre RPPA Core Facility, funded by NCI #CA16672, for producing the RPPA data included in the current work. Cell lines Co115, Colo320, EB, FRI, HT29, IS1, IS3, LS1034, LS174T, TC71, SW480 and V9P were kindly provided by Dr. Richard Hamelin (INSERM and UPMC, France), and CaCo2 and DLD-1 from PhD Juha Rantala (Oregon Health and Science University, USA).

The study was supported by the Norwegian Cancer Society (project number 72190-PR-2006-0442 and 6,824,048–2016), Southern and Eastern Norway Regional Health Authority, the Research Council of Norway ( FRIPRO Toppforsk , project number 250993 and Centres of Excellence funding scheme, project number 179571) and the foundation Stiftelsen Kristian Gerhard Jebsen .

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Gene expression data is available from NCBI’s Gene Expression Omnibus with accession number GSE97023. Remaining rawdata is available from authors upon reasonable request.

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Conceived the study: RAL. Study design: KCGB, PWE, RIS, OM, AS, RAL. Acquired data: KCGB, PWE, IAE, BJ, JB, SAD, MB, ME, LAMZ, GEL. Analyzed and/or interpreted data: KCGB, PWE, AS, RAL, GEL. Drafted the manuscript: KCGB, PWE, AS, RAL. Revised and approved the manuscript: all authors.

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Additional files

Additional file 1: tables s1-s14..

Supplementary tables. (XLSX 4615 kb)

Additional file 2: Figure S1.

DNA level aberrations. a SNVs and indel counts in 34 cell lines. MSI cell lines generally displayed numerous SNVs/indels, in contrast to MSS cell lines, although DLD-1/HCT15 were less typical with a lower indel burden compared to remaining MSIs. b The percentage of the genome with aberrant CNA reflects MSI status rather than CMS subtype. The figure includes 29 unique MSI/MSS cell lines. c CMS frequency of CNAs. Vertical axis indicates frequency, horizontal axes shows chromosomes 1–22, separated by vertical lines (whole lines separates chromosomes, dashed lines separates chromosome arms). The most common gains in CMS2 (5 or more out of 9 CMS2 MSI/MSS cell lines) were found on 3q, 8q, 13q, 17q, 20p and 20q, while regions of loss were frequent on 1p, 3p, 4q, 6p, 6q, 8p, 16p, 16q, 17p, 18 p, 18q, 20p and 22q. In CMS4 the most common gains (4 or more out of 7 CMS4 MSI/MSS cell lines) were found on 3q, 5p, 5q, 7p, 7q, 12p, 20p, 20q and 22q, while losses were frequent on 3p, 4p, 4q, 6q, 15q, 17p, 18q and 22q. The plots for CMS2 and CMS4 are placed together for easier visual comparison. A frequency plot for CMS3 was included, but the low sample number limits interpretations of frequent alterations in this group. d Differential frequencies of CNAs in undifferentiated versus colon-like cell lines. The vertical axis indicates the frequency difference between undifferentiated – colon-like cell lines (i.e. the frequency in undifferentiated cell lines minus the frequency of aberration in colon-like cell lines). The horizontal axis indicates chromosomes 1–22 (chromosomes separated by whole lines, chromosome arms separated by dashed lines). Yellow areas represent regions with higher frequencies of CNAs in colon-like cell lines, purple areas represent regions with higher frequencies of CNAs in undifferentiated cell lines. CMS: consensus molecular subtype, CNA: copy number aberration, MSI: microsatellite instable, MSS: microsatellite stable, SNV: single nucleotide variant. (PDF 830 kb)

Additional file 3: Figure S2.

Expression differences between colon-like and undifferentiated cell lines. a PCA plots show the spontaneous split between the two subgroups in all three datasets (mRNA, miRNA and protein). b Volcano plots show differentially expressed genes in undifferentiated ( blue ) versus colon-like ( yellow ) cell lines on the mRNA, miRNA and protein levels. Horizontal dashed lines mark the highest p -value that produces an adjusted p -value of <0.01. Vertical dashed lines mark log 2 fold-change (1 for mRNA/miRNA, 0.1 for protein). The top five differentially expressed mRNA/miRNA/proteins in terms of log 2 fold-change within these thresholds are indicated by names, and the rest by filled circles. PCA: principal component analysis, PC1: principal component 1, PC2: principal component 2. (PDF 1095 kb)

Additional file 4: Figure S3.

Outlier analysis reveals high level amplification events associated with marked mRNA expression changes, pointing to potential driver genes in individual cell lines. a A total of 280 genes were nominated, figure shows 15 nominated genes overlapping with the COSMIC Cancer Gene Census. Vertical axis shows gene expression values (log 2 scale) and horizontal axis shows copy number aberration values (PCF values, log 2 scale). The analysis pinpointed CNA values that were substantially higher in one or a few cell lines compared to remaining cell lines and hence 14 of the nominated genes were high amplification events, while one gene (SS18) was nominated on basis of all samples having loss except V9P. b Ten outlier genes had available associated protein data and for most genes the increase in gene expression was consistent on the protein level. Vertical axis shows relative protein expression (median centered normalized log 2 values), horizontal axis shows gene expression values (log 2 scale). (PDF 1233 kb)

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Berg, K.C.G., Eide, P.W., Eilertsen, I.A. et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol Cancer 16 , 116 (2017). https://doi.org/10.1186/s12943-017-0691-y

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DOI : https://doi.org/10.1186/s12943-017-0691-y

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Guide for Selection of Relevant Cell Lines During the Evaluation of new Anti-Cancer Compounds

Affiliations.

1 Departamento de Farmacologia, Facultad de Medicina, Universidad Nacional Autonoma de Mexico (UNAM), Ciudad de Mexico, Mexico.
2 Unidad Periférica de Investigación en Biomedicina Traslacional, Facultad de Medicina, UNAM, Ciudad de Mexico, Mexico.
3 Doctorado en Ciencias Biomédicas, UNAM, Ciudad de Mexico, Mexico.
4 Drug Design Group, Department of Pharmacy, University of Groningen, Groningen, Netherlands.
5 Unidad de Investigacion Medica en Enfermedades Oncologicas, Centro Medico Nacional SXXI, IMSS. Ciudad de Mexico, Mexico.
6 Posgrado en Ciencias Biológicas, UNAM, Ciudad de Mexico, Mexico.
7 Departamento de Fisiología, Facultad de Medicina, UNAM, Ciudad de Mexico, Mexico.
PMID: 29697026
DOI: 10.2174/1871520618666180220120544

Background: Human cancer cell lines are valuable models for anti-cancer drug development. Although all cancer cells share common biological features, each cancer cell line has unique genotypic/ phenotypic characteristics that affect drug response. Thus, the information obtained with a specific cancer cell line cannot be easily extrapolated to other cancer cells. Consequently, cell line selection during experimental design is critical for providing proper and clinically relevant structure-activity analysis.

Methods: Herein, we critically review the use of cancer cell lines as tools for activity analysis by comparing two different scenarios: i) the use of multiple cancer cell lines, with the NCI-60 Program as the most representative example; and, ii) the selection of a single cell line with specific biological characteristics that match the rationale of compound design.

Results: Considering that most laboratories evaluate the activity of new compounds using few cell lines, we provide a systematic strategy for selection based on the expression levels and genetic status of the target and the effectiveness of target inhibition or silencing. We exemplify the use of public databases for data retrieval and analysis as well as the critical comparison of such information with published results.

Conclusion: This approach refines cell line selection, avoiding the perpetuation of published poor selection and enhancing the relevance of the results.

Keywords: Anti-cancer drugs; CD44; EGFR; NCI-60; cancer cell line; cancer genomics; preclinical screening..

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LaNts and Laminins

The Hamill lab blog

Cell lines / cell culture in your methods sections

Required details;

Media formulations and suppliers information including all supplements
Seeding densities for specific experimental procedures (cells per flask or dish size)
Times from seeding to experiment
Plasticware supplier
Description of any substrate treatment – coating protein name, conc, supplier, coating approach
Independent experimental unit level (see * below) – how many “biological repeats” how many “technical repeats” per experiment.

In addition;

Immortalised/permanent lines;

Line name, species, origin tissue, type.
Supplier (commercial source details or collaborator)
Reference(s) for first description/characterisation
How you validated them; genetic fingerprinting? blotting/immunofluorescence/flow etc (usually the data associated with validation goes in supplementary figures)
Passage number, feeding schedule, etc
Immortalisation technique (SV40T, E6/E7, hTert etc)

Primary isolated cells

Source tissue / supplier
Number of donors and donor characteristics (age, sex, disease status etc)
Mycoplasma screening approach (kit, primer and PCR details)
Validation – images/blots/flow data etc. References to support these choices. See also the antibodies section for information associated with any antibody based validation

Note; for PhD thesis/dissertations – the isolation/establishment of a new primary cells or immortalised line might be quite an involved process that is integral to your data and so could end up as figures in your main results rather than as supplemental.

*Stats comment – Identifying the independent experimental unit

Hopefully by the time you are writing a methods section someone has had a proper chat/lesson about experimental design…!

Basically, cell culture experiments often get described as “technical replicates” or “biological repeats” however, what is what, and what is appropriate for your studies is something that you have decided during your experimental design and analysis. It is not obvious to the reader! So that means you should tell them.

Simply the “biological repeats” generate the numbers you actually use in your analysis. The number of biological repeats you have is your n number. Depending on the experiment, this is usually 1 donor = 1 biological repeat for primary cells and either 1 independently thawed population of immortalised cells or one separately passage flask of cells – this decision depends on your viewpoint and where you think non-independence comes in.

Technical replicates are the multiple points that you gained from within your experiment; these are used to generate the value for the biological repeat. Eg if you have 3 wells of control cells and 3 wells of drug treated cells that you ran together in a single experiment, your “biological repeat” would be 1, your technical replicates would be 3.

Go back to methods tips pages (+ links to examples of other sections)

Go back to the full methods guide (+ links to examples of other sections)

Guidelines for the use of cell lines in biomedical research

R J Geraghty 1 ,
A Capes-Davis 2 ,
J M Davis 3 ,
J Downward 4 ,
R I Freshney 5 ,
I Knezevic 6 ,
R Lovell-Badge 7 ,
J R W Masters 8 ,
J Meredith 9 ,
G N Stacey 10 ,
P Thraves 11 &

British Journal of Cancer volume 111 , pages 1021–1046 ( 2014 ) Cite this article

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Cell culture
Health policy
Medical research

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

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An efficient low cost means of biophysical gene transfection in primary cells

Introduction.

Problems associated with cell culture, such as cell line misidentification, contamination with mycoplasma and genotypic and phenotypic instability, are frequently ignored by the research community. With depressing regularity, scientific data have to be retracted or modified because of misidentification of cell lines. Occult contamination with microorganisms (especially mycoplasma) and phenotypic drift due to serial transfer between laboratories are frequently encountered. Whatever the nature of the cell culture operation, large or small, academic or commercial, such problems can occur. The aim of these guidelines, updated from the previous edition of 1999, subsequently published in the British Journal of Cancer ( UKCCCR, 2000 ), is to highlight these problems and provide recommendations as to how they may be identified, avoided or, where possible, eliminated.

Many countries now have legislation and Codes of Practice governing the use of human and animal tissue samples for research applications and these guidelines highlight the main legal and ethical issues that may be encountered.

The guidelines, prepared during 2013 by an ad hoc committee sponsored by Cancer Research UK, are meant to provide a series of pertinent and accessible reminders, which should be of benefit both to those for whom using cell lines is a new skill and those who may, despite years of experience, have allowed suboptimal procedures to become part of local practice. The guidelines are not meant to substitute for the many excellent textbooks that provide detailed information on many aspects of cell culture techniques and procedures. The guidelines are directed mainly at scientists in the UK but the principles will have international application.

Definitions of some terms frequently used in tissue culture are given in Box 1 .

Any references to commercial products are given for information only and no product endorsement is intended or implied.

Box 1 Definitions of terms frequently used in tissue culture

Also refer to the studies by Schaeffer (1990) and Freshney (2010) .

Anchorage dependence : the requirement for attachment in order for cells to proliferate.

Anchorage independence : the ability of cells to proliferate in suspension, either stirred or suspended in agar or Methocel.

Authentication : corroboration of the identity of a cell line with reference to its origin.

Cell cloning : see ‘ Cloning ’.

Cell concentration : number of cells per ml of medium.

Cell density : number of cells per cm 2 of growth surface.

Cell line : the progeny of a primary culture when it is subcultured. A cell line may be finite ( qv ) or continuous ( qv ).

Cell strains : cell lines that have been purified by physical separation, selection or cloning, and which have specific defined characteristics, for example, BHK-21-PyY, anchorage-independent cells cloned from the BHK-21 cell line following transformation with polyoma virus.

Cloning : the generation of a colony from a single cell; subculture of such a colony would give rise to a cell strain. Because of potential confusion with molecular cloning, this term is probably better modified to ‘ Cell cloning ’.

Confluence : a cell density at which all cells are in contact with no remaining growth surface.

Contact inhibition : strictly, the loss of plasma membrane ruffling and cell motility on contact in confluent cultures, but often used to imply loss of cell proliferation after confluence, better referred to as ‘ Density limitation of cell proliferation ’.

Continuous cell line : a cell line with an indefinite lifespan (immortal, over 100 population doublings; see also Immortalisation ).

Density limitation of cell proliferation : the reduction or cessation of cell proliferation at high cell density.

Differentiation : acquisition of properties characteristic of the fully functional cell in vivo .

DNA profiling : the assay of hypervariable regions of satellite DNA, usually by determining the frequency of short tandem repeats in microsatellite DNA using PCR of individual loci with defined primers.

Established cell line : the use of this term is discouraged because it is ambiguous; the preferred term is continuous cell line ( qv ).

Explantation : isolation of tissue for maintenance in vitro .

Finite cell line : a cell line that survives for a fixed number of population doublings, usually ∼ 40–60, before senescing and ceasing proliferation.

Generation number : the number of population doublings of a cell line since isolation.

Growth curve : a plot of cell number on a log scale against time on a linear scale.

Immortalisation : the indefinite extension of lifespan in culture, usually achieved by genetic modification, but already acquired by some cancer cells.

Passage : the event of subculture ( qv ), used to define the number of subcultures that a cell line has gone through since isolation. If used of continuous cell lines more usually the number of subcultures since last thawed from storage.

Primary culture : a culture from the time of isolation until its first subculture.

Primary explant : a small cellular fragment removed from tissue and placed in culture.

Provenance : details of the origin and life history of a cell line including various accidental and deliberate manipulations that may have a significant effect on its properties, latent or expressed.

Split ratio : the amount by which a culture is diluted before reseeding, usually a whole number.

Subculture : the transfer of cells from one culture vessel to another by dissociation from the substrate if a monolayer, or by dilution if grown in suspension.

Transformation : a heritable change involving an alteration in the genotype, usually subsequent to immortalisation. It is best reserved to describe an alteration in growth characteristics associated with malignancy ( anchorage independence , loss of contact inhibition and density limitation of cell proliferation , and tumorigenesis in vivo ).

Tumorigenesis : formation of a tumour in vivo , in the current context from implanted cells or tissue.

1. Cell line development, acquisition and authentication

Section 1. summary.

Record all data relevant to the origin of the tissue when starting a new cell line and keep tissue for DNA profiling.

Make sure the names of new cell lines are unique.

Acquired cell lines should come from a reliable source and must be authenticated to avoid misidentification.

Authenticated cells should be banked for future use and cultures replaced regularly from frozen stock.

Regulations often apply to the distribution of cell lines and only authenticated stocks should be distributed.

Cell lines can be developed in-house, acquired from other laboratories (if there is no more reliable source) or purchased from a cell bank. Whatever the source, it is essential to ensure that the cells are authenticated and free from contamination such as mycoplasma.

1.1. Developing a new cell line

Deriving a new cell line, especially when human, from fresh tissue is an expensive and time-consuming exercise. The subsequent value of the new cell line will depend on the ability to authenticate its origin and on the associated information that is available.

1.1.1. Tissue

In addition to tissue taken for culture, if donor or patient consent and/or ethical review permit (see Section 2.1), it is recommended that additional material is stored for:

Confirmation of origin (authentication) (see Section 1.2.2). A small portion of the sample used for primary culture (or a blood sample or DNA derived from the donor) should be frozen or processed immediately. The tissue or DNA can then be used to demonstrate unequivocally that the cell line is derived from the putative donor. Short tandem repeat (STR) profiling is a recommended method for the purpose of authentication, although additional information on genotype (karyotype, copy number variation (CNV) mapping, or even whole-genome sequence) will sometimes help ensure identity.

Histopathological confirmation. A small portion of the sample being used to originate the culture should be fixed in formalin and used for histopathological assessment, ideally by the same histopathologist reporting the surgical specimen if this is from a patient. This step is particularly important if a patient sample is supplied to the laboratory directly by a clinician, because it may not be representative of the surgical specimen sent to the histopathologist. For instance, it may be taken at some distance from a tumour and consequently lack cancer cells, or it may be from a region that is unaffected by a specific pathology caused by a genetic or epigenetic defect.

Normal tissue for comparison. A small quantity of blood (e.g., 10 ml) or normal tissue should be frozen. This tissue can later be used to look for genetic differences and could also be used for authentication. In the case of iPSC lines, or when direct reprogramming is used to derive one somatic cell type from another, it is also good practice to cryopreserve stocks of the original cells used. These could be important to derive additional cell lines using new reprogramming technology, but also to provide original donor material for validation of later discoveries made using the cell line. If somatic cell nuclear transfer (SCNT) or ‘cloning’ technology is used to derive cell lines, such as ES cells, then cells or tissue from both the somatic cell donor and oocyte donor should be kept in order to match nuclear and mitochondrial DNA, respectively.

1.1.2. Clinical information

If donor or patient consent and ethical reviews permit (see Section 2.1 and Box 2 ), as much of the following information as possible should be recorded and stored securely:

Age and sex of donor/patient.

Hospital and pathology numbers.

Site of origin and nature of tissue specimen.

Stage and grade of cancer or other syndrome, or pathology.

Copy of histopathology report.

Clinical history including treatment.

Additional information such as tumour marker status, genetic information and family data etc.

Evidence of informed consent and waiver of commercial rights by donor.

Information that could be used to unambiguously identify the donor – including name, hospital number, address and date of birth – should be stored with a higher level of security, preferably separate from other data. In the UK an NHS contract or honorary contract will be required to access patient details and such information should never be shared with unauthorised colleagues or released into the public domain.

1.1.3. Accessory information

The more information that is kept regarding the origin and derivation of the cell line, the more likely it is that the cell line will be useful for the originator and any subsequent users. New cell lines should be characterised to confirm their immortality, authenticity and tissue or cell type ( Drexler and Matsuo, 1999 ).

It is recommended that a complete record of the culture details are kept at initiation or receipt and during all subsequent manipulations, particularly up to the point when the cell line is banked in liquid nitrogen (LN 2 ) (see Section 1.4.1). This should include the type, sources and batch numbers of all media and additives and the methods by which the cell line was established. It is helpful to record the split ratios and the passage number.

Although it may be necessary to use antibiotics in the primary culture, they should be removed as soon as possible and the cells tested for mycoplasma (see Section 4.2.4 and Table 3). The type of assay used for mycoplasma detection should be stated, as should the frequency and date of the last test.

Images of the primary culture, early passages and some later passages should be stored for publication and future reference.

If a cell line is genetically modified (including methods used to achieve immortality when relevant), it is essential to describe the process used, including details of sequences, mode of insertion and antibiotic resistance markers. Additional tests may be necessary to demonstrate lack of infectivity, for example, following transduction using lentiviral or retroviral vectors. For hybridomas, details of the sources of both sets of cells are needed. Where animal tissue is used to originate a culture, it is important to record the species and strain, age, sex and genetic status.

For iPSCs, or cell lines derived by direct reprogramming, the methods used should be described, including the genes and vectors used, whether these are integrating, inducible, episomal or excisable, or whether ‘small molecule’ chromatin-modifying drugs, shRNA, or other reprogramming methods have been included. This is necessary as the reprogramming method may affect the properties of the cells and may be important for comparisons with other cell lines.

1.1.4. Cell line designation

It is essential that the designation of the cell line is unambiguous, unique and maintains donor anonymity ( Freshney, 2010 ). The format could be as follows: Institution – Source or series – code or log number – clone number; for example, MOG-G123-D4 (Medical Oncology Glasgow – Glioma Cell line 123 – clone D4). The full designation should be used in the materials section of publications. A similar scheme has been proposed for induced pluripotent stem cell (iPSC) lines ( Luong et al, 2011 ).

If the cell line is obtained from another source, its original designation must be retained. If obtained from a cell bank, its accession number should be quoted in publications. Genetic modifications, sublines and clones should be indicated by a suffix, following the original designation. It is important that the designation is unique so that there is no ambiguity with other cell lines or biological resources during literature searches (a simple search in PubMed will confirm this).

1.1.5. Publication

The first publication should include the information described in the previous sections and subsequent publications should cite the first publication. Every publication should confirm that the cultures have been tested for mycoplasma (see Table 3 and Section 4.2.4) and that the test is negative. It is possible to eliminate at least some types of mycoplasma from cell lines, although this would only be worth attempting for particularly valuable or unique cells. The first publication should also provide evidence that the cells have been derived from the individual claimed to be the source, with subsequent publications comparing stocks of that cell line to the STR profile or other evidence cited within the first publication. Some journals insist on cell lines being made available as a condition of publication, so that other laboratories can repeat the work. Some funding agencies and institutions also encourage or insist that cell lines derived with their support are made available to others, free or at cost, even if they also require an MTA. Information on deposits in cell banks or whom to contact to obtain cells is helpful in this regard. Publication of work with the cell line implies its entry into the public domain and the right of others to acquire the cell line from the originator or the nominated cell bank.

1.2. Acquiring a cell line from another laboratory

Acquisition of cell lines presents a number of potential hazards; cell lines may simply not be what they are claimed to be and a published description of a cell line with a certain property is no guarantee that it is still the same line or has that same property. The more laboratories that a cell line has passed through since its origin, characterisation and contamination testing, the less reliance should be placed on its documented properties. However, even the originator as a source is not a guarantee of authenticity. If the receiving laboratory wishes to place any reliance on historic data obtained with a cell line, it should always carry out its own testing procedures (see Sections 1.2.2 and 1.5.1) before accepting an incoming cell line into general use. An enormous amount of time, cost and effort can be wasted by scientists using cell lines that are either misidentified or contaminated.

The cell bank or laboratory of origin should be able to provide a certificated DNA STR profile for human cell lines and evidence of authentication using an appropriate technique for non-human cell lines (see Sections 1.2.2 and 1.5.1). However, the publication of full STR profiles for human cell lines from tissue donated anonymously may present ethical problems. While profiles of long-established cell lines have been made widely available, the profiles of recently isolated cell lines could potentially be used to re-identify the donor or their family. Guidance on managing such scientific data is given in the study by Isasi et al (2012) .

The name of the cell line should be checked against the International Cell Line Authentication Committee (ICLAC) database of misidentified cell lines ( ICLAC, 2013a ). The STR profile should be repeated at the time of banking the new cell line in LN 2 .

Human cell lines may carry pathogens, including viral contamination, representing a potential health hazard to laboratory workers (see Section 3.1). They may also become contaminated with bacteria, fungi, mycoplasma or viruses, which may spread to other cell lines. These contaminants may also be potential pathogens. If the cells are to be used in animals, whether as grafts of normal tissue or to derive tumours, or to make chimeras, it is also critical that they are tested and shown to be free of relevant pathogens, which might otherwise harm the animal colony or those who care for the animals. The cells or their derivatives may also be re-isolated from the animals for further study in vitro , in which case they need to be treated as a new sub-line and subject to further characterisation for genetic status as well as mycoplasma and other pathogens. Human cells passaged through animals could in theory have acquired replication-competent retroviruses from the animal host that could subsequently infect human cells, although the risk of this and of them being pathogenic to humans is very low.

In sourcing a cell line the establishment of provenance for that cell line should be a key requirement. This includes records of its origin and history, and quality control (QC) testing performed to ensure that it is free of contaminants ( Freshney, 2002 ). Cell lines should only be obtained from sources where this provenance is clearly documented.

1.2.1. Quarantine

New cell lines should be quarantined in the laboratory and in storage until their origin has been authenticated (see Section 1.2.2) and they are shown to be free of microorganisms (see Section 4.2.4 and Table 3). Ideally, a separate quarantine laboratory should be available for this purpose. The next best approach is to have a Class II microbiological safety cabinet (MSC) and an incubator dedicated for quarantine. If this is not possible, other steps should be taken to minimise the risk of spreading contamination, including (a) cells in quarantine should be handled only after all other cell culture has been completed that day, (b) the new cultures should be placed in a dedicated incubator or into a sealed container before going into a general incubator, (c) the MSC should be cleaned after use with a suitable non-corrosive disinfectant (see Table 4) and run for at least another 5 min before shut down.

1.2.2. Authentication

On receipt and before freezing a master cell bank (MCB) or seed stock (see Section 1.4), cell line authentication should be performed using an approved DNA-based method (see Sections 1.2.2 and 1.5.1) for confirming the origin of a cell line ( American National Standards Institute, 2011 ; ICLAC, 2013b ) and to check for misidentification. Ideally, it will be possible to compare the DNA with that of the tissue or donor of origin (see Section 1.1.1), but unfortunately this is only possible in a minority of the cell lines already available. Nevertheless, it is desirable that a STR profile is defined before the cell line is used, so that at least it can be distinguished from other cell lines in the same laboratory and shown to be unique with reference to an international database ( NCBI, 2013 ) or by contacting a reputable cell bank (see Table 1 ). It can then be tracked through subsequent transfers. For cell lines derived from inbred strains of mice, where STR profiles may not distinguish one line from another, but where a specific genetic alteration (mutation or transgene) has been introduced, a specific test for the allele in question should be established.

1.2.3. Characterisation

The user should also confirm that the cell line they obtain is fit for their own particular purpose. Even if a cell line is shown to be authentic, it may have lost a particular key characteristic with prolonged passaging. Karyotyping is a simple test that can reveal changes in a cell line. Indeed, it is routine to show that a line of ES cells or iPSCs has a normal karyotype if they are to be used for experiments involving production of chimeras and germ line transmission. Molecular assays to look for CNV or RNA profiling will also be indicative of changes, but are more costly. Nevertheless, a great deal of time and effort can be saved by confirming the appropriate characteristics before commencing work. It is also advisable to capture an image of the cell line in culture at different cell population densities and perform basic characterisation (e.g., calculating the population doubling time for that cell line) soon after arrival.

With newly developed cell lines it will also be important to confirm which type of cell the cell line is derived from using, for example, intermediate filament proteins, such as cytokeratins for epithelial cells, or specific cell surface markers, such as A2B5 for glial cells, and special properties required for the proposed study. More than one marker will be required for reliable characterisation.

1.3. Cell banks

A number of culture collections or cell banks have been established by either academic or commercial bodies (see Table 1 ). Cell lines from these sources are tested for identity and contaminating microorganisms that commonly occur in culture, so they are unlikely to be contaminated or misidentified, unless so stated in the accompanying literature. However, some of these cell lines have been acquired following multiple transfers between laboratories, so authenticity and freedom from microbial contamination are not guaranteed unless specifically stated in a Certificate of Analysis. The cell culture collections mentioned above routinely authenticate their cell line stocks and provide a Certificate of Analysis, including an STR profile, for each line they produce.

1.4. Storage and banking

Once a cell line has been developed or acquired and validated (i.e., shown to be authentic and uncontaminated) the first step to ensuring a reliable and reproducible supply of cells is the cryopreservation of about 20 1-ml ampoules, each containing 1–5 × 10 6 cells. This will provide the vast majority of laboratories a ready supply for many years. Depending on the size and duration of the operation it is often useful to have a tiered system: (a) an MCB or seed stock, containing 10–20 ampoules, which should be protected and not distributed; (b) a distribution stock generated from the seed stock, which is used to provide the end users with cultures ( Stacey and Doyle, 1997 ; Freshney, 2010 ) from which they will generate their own frozen stock. This user stock should contain sufficient ampoules to provide at least one ampoule for every 3 months of the proposed experimental period plus sufficient ampoules for contingencies; these cells should not be distributed other than to those within the group for whom they were frozen. Incorrect or serial banking (as occurs for cultures passed from one laboratory to another in a chain) results in a progressive increase in the population doubling number and additional risk of contamination or loss of key characteristics and to selection for abnormal growth characteristics accompanied by genetic and/or epigenetic changes.

1.4.1. Cryopreservation

Cell lines are preserved by freezing samples slowly (usually 1 °C min −1 ) in preservative (usually growth medium with 10% DMSO). An automatic controlled-rate cooling apparatus provides the most reproducible cryopreservation provided the freezing programme used has been optimised for that cell line’s requirements but simpler devices may suffice ( Freshney, 2010 ; Davis, 2011 ).

Certain cell types, for example, hESC, may require ultra-rapid freezing or vitrification ( Hunt, 2011 ) where water is frozen in situ to form a glass and not allowed to permeate out of the cell as in slow freezing and is often used to freeze stem cells.

Every time a batch of cells is frozen down, it is recommended that one vial is resuscitated immediately to check viability. Vials removed from the bank should be thawed rapidly (by immersion in a water bath at 37 °C) and the cell suspension diluted gradually with pre-warmed medium.

1.4.2. Storage

Cell stocks should be kept below −130 °C as viability may be progressively lost within a few months at −80 °C. Once at their final storage temperature it is also detrimental to warm them to −80 °C even for short periods. However, cells can be kept at −80 °C during the freezing process either for convenience, although usually for no more than a few days before being transferred to the definitive storage vessel, or when cells need to be kept frozen in multiwell dishes while waiting for results from a screen. This is commonly used during gene-targeting experiments with ES cells where it is necessary to screen many individual clones to find the relatively few that will be thawed for further growth and research. The multiwell dishes need to be thoroughly sealed so that they do not dry out at −80 °C.

Potentially infectious material must be stored in the vapour phase of LN 2 to reduce the risk of transfer of contaminating organisms ( Tedder et al, 1995 ). It also eliminates the hazard of LN 2 -penetrating ampoules that may then explode on warming. Storage in vapour phase of LN 2 is increasingly the norm for safety purposes but requires careful monitoring of the level of the LN 2 as the smaller volumes used in vapour-phase storage will run out quicker.

For security, important material, (e.g., MCBs) should be divided into more than one storage vessel, preferably on different sites. Deposition and removal of frozen stocks should be recorded and controlled to avoid loss of entire stocks and to indicate when re-banking of stocks should be performed. Labelling of frozen stocks should be legible and resistant to LN 2 . It is recommended that the label on the frozen vial should contain the name of the cell line, batch number and freeze date as a minimum. These labels should be printed rather than handwritten, using labels that are suitable for prolonged storage in liquid nitrogen. Barcoding has proved to be a simple method that can contain most information on a small label.

The location of the vials should be detailed in a spreadsheet or database linked to details of the origin and characteristics of the cell line and the QC measures that have been applied to it.

Hazards associated with the use of LN 2 include frostbite and cold burns, asphyxiation (i.e., oxygen depletion) and risk of infection and injury due to explosion of ampoules (see Section 3.1).

Cryostorage vessels should be fitted with alarms and storage temperatures checked regularly. It is recommended that levels of LN 2 in the storage vessels are recorded at least once a week. Periodic audits for evidence of regular maintenance, monitoring and stock control will also help ensure safety and security of storage facilities.

1.5. Cell line misidentification

Misidentification occurs as a result of cross-contamination, poorly controlled manipulation or clerical error and implies a failure in good cell culture practice (GCCP); for example, accidental transfer of cells to a stock bottle of medium, having two cell lines in an MSC at the same time, mislabelling a flask or ampoule, or thawing the wrong ampoule. Other sources of cross-contamination are if feeder cells (e.g., as often used in ES cell culture) are still mitotically active due to inadequate irradiation or treatment with mitomycin C, or if conditioned media is prepared without adequate filtration to remove cells. Whenever a rapidly growing, continuous cell line is maintained in a laboratory there is a risk that it may cross-contaminate (i.e., replace) other, more slowly growing lines. There is a long history of this problem, highlighted in the 1960s and 1970s ( Gartler, 1967 ; Nelson-Rees and Flandermeyer, 1976 ; Nelson-Rees and Flandermeyer, 1977), but now often ignored. Few authors using cell lines such as KB, Int-407, WISH, Chang liver or Hep-2 acknowledge that they are in reality working with HeLa cells. Similarly, some cell lines with a variety of names and claimed tissues of origin are in fact MCF-7 (breast cancer) or T24 (bladder cancer) cells. Whatever the purpose of the experiments, it is essential to know the derivation of the cells. Even if the process being studied is not cell type-specific, others may cite the work in a context where it is.

Changes in cell behaviour or morphology may indicate cross-contamination and constant vigilance and attention to GCCP are essential (see Section 1.5.1).

A list of known misidentified cell lines is available from ICLAC ( ICLAC, 2013a ). However, even if a cell line is not on that list, a laboratory should always test to ensure that its own stocks of that cell line are authentic.

Simple precautions must be taken to minimise the possibility of misidentification, including:

All culture vessels must be carefully and correctly labelled (including full name of cell line, passage number and date of transfer), as must storage containers.

Only one cell line should be used in an MSC at any one time. After removal of the cells, the cabinet should be swabbed down with a suitable liquid disinfectant and run for a minimum of 5 min before the introduction of another cell line.

Bottles or aliquots of medium should be dedicated for use with only one cell line.

The formation of aerosols must be kept to a minimum.

A return to frozen stocks should be made regularly (except where essential, never grow a cell line continuously for >3 months or 10 passages, whichever is the shorter period, unless otherwise validated).

1.5.1. Recognising cell line misidentification

Short tandem repeat profiling is the standard method for authenticating cell lines. An American Standard (ASN-0002 2011) provides information on how to use STR profiling for the authentication of human cell lines. Recommendations from the standard should be followed, including the use of at least eight core STR loci and application of match criteria (80% match threshold) to allow for a small amount of genetic drift in some cell lines. The standard can be purchased at the ANSI eStore ( American National Standards Institute, 2011 ). Large numbers of organisations offer STR profiling of cell lines at a small cost.

For non-human cell lines, best practice will vary with the species being tested. As a minimum, it is recommended that non-human cell lines are tested for species specificity. Appropriate test methods include karyotyping ( MacLeod and Drexler, 2005 ), isoenzyme analysis ( Freshney, 2010 ) and mitochondrial DNA typing (DNA barcoding) ( Cooper et al, 2007 ; O'Donoghue et al, 2011 ). It is also possible to compare partial sequences with the full genomic sequences that now exist for a number of human cell lines, including cancer cells, and for several inbred mouse lines and other commonly used species from which cell lines have been derived. A valuable resource is the Ensembl sequence database, a joint project between the European Molecular Biology Laboratory and the Wellcome Trust Sanger Institute ( Birney, 2004 ).

1.6. Cell line distribution

1.6.1. introduction.

Transferring a cell line between laboratories may involve transport within a city, country or between continents. Therefore, consideration will have to be given to the condition of the cells, the means of transport and the legal requirements (see Section 2.1.7). Cell lines may be transported either as growing cultures or as vials of frozen cells.

Within the UK and European Union, use of a courier service should ensure delivery within 48 h to most destinations. Delivery to most places outside of the European Union should be possible within 96 h and this is compatible with sending growing cultures. However, it is impossible to guarantee that packages have remained under appropriate conditions (e.g., temperature, vibration-free) throughout the transport period. If frozen vials are sent, the fact that the refrigerant remains within the package on receipt should be sufficient to ensure that transport conditions have been acceptable.

Some couriers will not accept boxes containing solid carbon dioxide or LN 2 for transportation; therefore, a specialist courier may need to be appointed.

1.6.2. Transport containers

Cultures of adherent cells growing in flasks should be sent with the flask filled completely with medium at the correct pH. Disadvantages of this procedure are that the flask is heavy; there is a considerable volume of medium to leak if the flask is broken and cultures may subsequently become infected because of medium around the neck and cap of the flask. An alternative method is to remove all except a few drops of medium from the flask, gas appropriately, and seal the flask. The small volume of medium is sufficient to keep the cells moist but insufficient to allow frothing to occur and cells can remain viable for at least 72 h if kept cool.

For suspension cultures or cells that grow as floating aggregates, 2-ml plastic freezing vials are suitable containers for transport. Cells in medium should be transferred to the vial in a volume of 1.0–1.5 ml and medium then added drop-by-drop to fill the vial before replacing the screw cap. Because of their size, such vials can be sent in small padded envelopes if suitably sealed in a plastic bag or secondary container containing sufficient absorbent material to soak up the medium in the event of a breakage or leak.

Insulated boxes suitable for transport of frozen vials of cells are used by various laboratory supply companies for distribution of frozen reagents. Such boxes typically have 5-cm-thick walls with a central cavity of 15 × 15 × 15 cm. This can be filled with solid CO 2 , which will maintain temperature for a maximum of 4 days. Always have a vent for boxes holding solid carbon dioxide to allow gas to escape and make sure that the vials of cells are well sealed or in a gas-tight container to avoid CO 2 gas entering, because this can significantly lower the pH of the medium. Use appropriate signage on the outside of the package, for example, UN1840 for dry ice (solid CO 2 ).

An alternative that is more expensive to buy and use, but which is very reliable, and increasingly used to send valuable samples, such as frozen embryos, is a Dry Shipper. These are specially designed Dewar’s for liquid nitrogen, but where this is contained within a cryo-absorbant material. There is therefore no risk of spillage and cell vials (or straws) are kept frozen in the vapour phase at stable temperatures below −150 °C for up to 10 days, depending on the size, capacity and make of the dry shipper. It is recommended to use makes and models that conform to IATA shipping regulations.

1.6.3. Practicalities

Experience dictates that adherence to the following points will increase the probability of successful transfer:

Communicate fully with the carrier and the recipient in advance. Ensure that they both know the collection time and the anticipated delivery time. Exchange contact details in case problems arise. Keep shipping reference numbers such as Airway Bill Numbers and share them with the receiver of the package in case of delays or misrouting.

Inform the recipient of what type of containers are being sent and the state of the cells and provide details of what to do with the cells when they arrive, to ensure that they have the correct medium available and that they are familiar with the growth characteristics of the cells.

Ask the recipient to notify you when the cells arrive or when the cells have failed to arrive within a reasonable period.

Send packages on a Monday to improve the chance of a weekday delivery.

Ask the recipient to establish, as a high priority, their own frozen stock of the cells so that repeated transport is not needed.

1.7. Regulations for the transport of cells

Various regulations must be complied with when sending cells to other laboratories. These include legal requirements of various countries and regulations established by individual carriers. It is strongly recommended that full details of these are obtained before any transport is attempted. Regulations concerning the transport of potentially dangerous goods are published by the International Air Transport Association ( IATA, 2013 ) and updated annually. There may also be issues of consent, with respect to use, distribution and export from specific countries, and the relevant documentation may be required by research funders and by journals.

In the case of human ESC lines there are special non-statutory regulations required under Medical Research Council (MRC) funding. These require that projects using hESC lines or their import, export or movement from one centre to another must have the approval of a national oversight body, the Steering Committee for the UK Stem Cell Bank (UKSCB) and for the Use of Stem Cell Lines ( MRC, 2013 ).

It is beyond the scope of these Guidelines to spell out in detail the full regulations. However, the following points may be useful in providing general guidance:

1.7.1. Within the UK

Use an approved National Carrier or Courier service.

1.7.2. Import to the UK

While there are few restrictions on the movement of cell cultures within the European Union, importation of certain animal cells from other countries into the UK requires a permit from the Department for Environment, Food and Rural Affairs ( DEFRA, 2013 ). This is particularly important for cells from agricultural species, including poultry, where there is a serious risk of importing non-endemic viruses.

Some countries are concerned about export of indigenous genetic resources, which could encompass tissues and cell lines, and may have imposed restrictions on export for any type of research, but especially for potentially commercial applications.

1.7.3. Export from the UK

Apart from the USA and Australia, few countries have specific regulations regarding the import of cell lines and hence sending cells abroad should not present major problems. However, if material is classified as Advisory Committee on Dangerous Pathogens category 2 or above ( ACDP, 2004 ), special conditions apply and the sender must undergo formal training. It is recommended that the cell line(s) are sent by courier service and that the contents of the package must be clearly labelled on the ‘shipper's declaration’ as ‘biological material for research purposes’. Include the contact details of the sender on the outside of the package in case of mishap/accident and check with the receiving laboratory and the courier so you know what is required before you proceed.

An application should be made for a Veterinary Permit from the US Department of Agriculture ( USDA, 2012 ) before shipment for the importation of cell lines or their products into the United States and to the Department of Agriculture, Fisheries and Forestry ( DAFF, 2013 ) for Australia. A copy of the permit should be taped to the outside of the package.

For some countries, it is not just the cell line that is relevant but also whether the medium contains serum and its source. Use of a serum replacement or serum-free medium can provide a simple solution.

2. Derivation of a new cell line

Section 2. summary.

There are ethical and legal requirements for obtaining tissue for cell lines.

Specific regulations apply to the use of human tissue for research purposes.

Patient consent is usually required for the use of human tissue samples and ownership must be defined.

Separate regulations may apply to initiating cell lines from animal tissues.

Transfer of cell lines from one laboratory to another may require a material-transfer agreement (MTA).

It is not the purpose of this document to describe the methodology for developing primary cultures and deriving cell lines from them, because extensive literature is already available. However, there are specific precautions and procedures that those proposing to do this type of work should be aware of.

2.1. UK legal and ethical requirements

These may be summarised as follows:

Research involving human tissue samples will require ethical approval. To this end the Human Tissue Act 2004 legislates on the use of human tissue samples for a number of scheduled purposes including research. Informed patient consent may be required to store and use human tissue samples for research purposes and a Human Tissue Authority licence may be required to store human tissue samples for research purposes. Once a human cell line is established it is no longer covered by the Act.

Any patient data where the patient name is recorded should be managed under the Caldicott Principles. These require the laboratory to have a Caldicott Guardian to assure compliance with these guidelines ( Caldicott, 2013 ).

The Human Fertilisation and Embryology Act 1990 (amended 2008) legislates on research using early human embryos up to 14 days of development or the first signs of primitive streak formation and is regulated and licensed by the Human Fertilisation and Embryology Authority (HFEA). The HFEA is not concerned with tissues from later-stage embryos or foetuses (e.g., from ectopic pregnancies or terminations) ( HFEA, 2008 ).

Clinical trials of cell-based medicinal products are regulated by the Medicines and Healthcare Products Regulatory Agency in compliance with the Medicines for Human Use (Clinical Trials) Regulations 2004 ( MHRA, 2004a ).

An MTA should accompany all transfers of created cell lines between organisations and should define specific details including ownership, intellectual property rights and patent rights.

The use of animals in experiments and testing is regulated under the Animals (Scientific Procedures) Act 1986 (ASPA). ASPA has now been revised to transpose European Directive 2010/63/EU on the protection of animals used for scientific purposes ( EU Directives, 2010 ) and the revised legislation came into force on 1 January 2013. ASPA is not directly relevant to the derivation of a cell line from an animal that has been killed (by a schedule 1 method). However, it is relevant if any regulated procedure is required, such as tissue biopsy of a live animal, administration of substances, or derivation of a genetically altered animal. It is also relevant if cells are to be introduced into a live-born animal or animal embryo. While for most experiments it will make little difference with respect to regulation under ASPA whether the cells are of animal or human origin, some involving the latter may be considered contentious, especially if they concern the reproductive system or have the potential to lead to human characteristics developing in an animal (see Academy of Medical Sciences Report on Animals Containing Human Material ( www.acmedsci.ac.uk )). New regulations and guidance on this type of research are being introduced and such experiments will be considered by the new Animals in Science Committee of the Home Office.

In the United States, information on human issues is available through the Presidential Commission for the Study of Bioethical Issues ( PCSBI, 2013 ) and the Office of Human Research Protections ( OHRP, 2011 ). Information on animal usage in the USA is available through the Office of Laboratory Animal Welfare ( OLAW, 2013 ).

2.1.1. Ethical approval for the use of human tissue in research

All research projects, studies and clinical trials conducted in the UK involving National Health Service (NHS) patients, human tissue samples and identifiable clinical data must be favourably reviewed by a recognised NHS Research Ethics Committee (REC) before they can proceed. The principal aim of the REC is to safeguard the rights, safety, dignity and well-being of individuals participating in research (see Box 2 ). These RECs are managed and administered through the National Research Ethics Service ( NRES, 2013 ), which is part of the NHS Health Research Authority ( NHS, 2013 ). All applications for ethical review by a REC must be made through the electronic Integrated Research Application System ( IRAS, 2013 ). In addition most NHS Trusts and Universities will have their own Research and Development Departments, which will approve all proposed new research, involving human tissue samples, before submission for ethical approval and scientists should make themselves familiar with their host organisation’s local rules and policies.

2.1.2. The Human Tissue Act 2004

The Human Tissue Act 2004 ( HT Act, 2004 ) came into force on 1st September 2006, covers England, Wales and Northern Ireland and established the Human Tissue Authority (HTA) to regulate activities concerning the removal, storage, use and disposal of human tissue samples for a number of defined Scheduled Purposes, including ‘research in connection with disorders, or the functioning of, the human body’. The HTA also licenses a number of activities including removal of relevant material from a deceased person and storage of relevant material for a Scheduled Purpose. Consent is the fundamental principle of the legislation. Different consent requirements apply when dealing with tissue from the deceased and the living. Scotland has separate legislation, the Human Tissue (Scotland) Act, 2006 . Both acts are broadly similar in principle, but the Scottish legislation is based on authorisation rather than consent ( Human Tissue (Scotland) Act, 2006 ).

2.1.3. Human cell lines and the Human Tissue Act 2004

The HT Act defines human tissue (‘relevant material’) as material that consists of, or includes, human cells. This includes blood, tissues and organs but does not include:

Material that contains no cells, for example, serum, plasma and urine (providing the urine is acellular).

Gametes (ova and sperm).

Material created outside of the body in vitro , for example, embryos and cell lines.

Therefore primary human tissue and cells (i.e., those removed directly from a person) are defined as relevant material under the HT Act. Cell lines derived from expansion of primary cell cultures in vitro are not relevant material, as all of the original cells will have divided and so the cell line has been created outside of the human body. The storage of cell lines created from primary human tissue, for research purposes, does not require an HTA licence and the use of such cell lines is not covered by the HT Act or regulated by the HTA. However obtaining, retention and storage of any of the primary material from which the cell line was derived will be subject to the HT Act and HTA regulation, as will any cell lines derived with the intention of use in human therapy under the HTA (2007) .

Under the HT Act consent is not required to store and use human tissue for research (including the creation of cell lines) if:

The tissue sample was obtained before 1 September 2006.

The tissue sample is from a living person and the proposed work is part of a research project or study approved by an NHS REC and the identity of the donor remains unknown to the researcher.

Under the HT Act a licence is not required to store human tissue for research if:

The tissue sample is being held for use in an ethically approved research project or study, or where approval is pending.

The tissue sample is being stored before transfer elsewhere, provided it is held for no longer than 1 week.

The tissue sample is being held while it is processed with the intention to extract components that are not relevant material and provided the processing does not take longer than 1 week.

The HTA has published a series of Codes of Practice that provide full guidance and lay down the standards expected for each of the Scheduled Purposes, in order to comply with the HT Act ( HTA, 2013 ). All scientists working in the UK who are producing cell lines from primary human material, for research purposes, should make themselves familiar with Codes 1 (Consent), 9 (Research) and 5 (Disposal).

2.1.4. Research using human embryonic stem cells

The Human Fertilisation and Embryology Act 1990 created the HFEA as an independent regulator of in vitro fertilisation (IVF) and human embryo research. One of the statutory functions of the HFEA is to license and monitor establishments undertaking human embryo research and this will include production of human embryonic stem cell (hESC) lines.

The original Act defined 5 purposes for which a research licence could be issued by the HFEA:

Promoting advances in the treatment of infertility.

Increasing knowledge about the causes of congenital disease.

Increasing knowledge about the causes of miscarriages.

Developing more effective techniques for contraception.

Developing methods for detecting the presence of gene or chromosome abnormalities in embryos before implantation.

Although these purposes did not preclude the derivation of human ESC, the reasons for doing so would have been limited by them. The Act had been passed before human ESC had first been derived, and there had been several other scientific advances, notably SCNT or cloning, which suggested that it needed updating. The Human Fertilisation and Embryology (Research Purposes) Regulations 2001 added three further purposes:

Increasing knowledge about the development of embryos.

Increasing knowledge about serious disease.

Enabling any such knowledge to be applied in developing treatments for serious disease ( HFEA, 2001 ).

The current version of the Act, passed in 2008, incorporates a number of very significant amendments, which were again made to accommodate rapid advances in science as well as changes in public attitudes and clinical practice ( Lovell-Badge, 2008 ). These amendments included provisions for research on ‘human admixed embryos’, including human embryos that have been altered by the introduction of one or more animal cells. This would include the generation of chimeras with human ESC or iPSC.

The HFEA has published a Code of Practice ( HFEA, 2013 ) that has a detailed section on Research and Training and lists all mandatory requirements for extraction, freezing, storage and use of human embryos, which are relevant to hESC research. In summary the HFEA can grant research licences for up to 3 years for individual, peer-reviewed research projects. All licence applications and renewals are evaluated by an HFEA Research Licence Committee. All new applications for a research licence must also have ethics approval (see Section 2.1.1). The HFEA charge an administration fee for granting and renewing project licences, which varies depending on the nature of the research. A requirement of the HFEA granting a research licence is that any cell lines produced must be deposited with the UKSCB ( UKSCB, 2013 ). All uses of hESC lines are subject to non-statutory regulation overseen by the Steering Committee for the UKSCB, which approves new research on hESC lines including their import, export and transfer between institutions. It also approves deposit and release of hESC lines for the UKSCB.

In response to the Human Fertilisation and Embryology (Research Purposes) Regulations 2001 ( HFEA, 2001 ) the UK’s MRC was required to put into place the Steering Committee for the UK Stem Cell Bank and for the Use of Stem Cell Lines. This committee oversees and approves the import, export, transfer and use of hESC lines within the UK and has also published a Code of Practice for the Use of Human Stem Cell Lines (2010) available via the MRC ( MRC, 2013 ).

Even though they are similar in properties and potential, human iPSC lines do not have to be deposited in the UKSCB, nor does the Steering Committee oversee their use. However, as with any other cell line, their use in a clinical setting would be regulated as described below.

2.1.5. The use of human cell lines as therapeutic agents

Research involving hESCs and other human tissue-derived cell lines will involve different regulatory authorities at different stages. For example, cell-based products that involve the destruction of a human embryo in their formation are initially licensed by the HFEA. Once an embryo has been disaggregated it is no longer subject to HFEA regulation. If the cells replicating from such a disaggregated embryo are intended for application on humans, they are then subject to the HTA (2007) up to the point of the first representative cell bank for that cell line. These regulations are administered by the HTA. However, hESC lines derived purely for research are not subject to this regulation. If the research project is to develop and manufacture a cell-based therapeutic product, then using the primary cells will remain under HTA regulation until the Medicines and Healthcare Products Regulatory Agency (MHRA) classifies the product as an Investigational Medicinal Product (IMP) or an Advanced Therapy Medicinal Product (ATMP).

In the UK clinical trials authorisation of all medicinal products is solely granted by the MHRA in compliance with the Medicines for Human Use (Clinical Trials) Regulations 2004 ( MHRA, 2004a ), which implement the EU Clinical Trials Directive 2001/20/EC ( MHRA, 2004b ). Favourable opinion from a recognised research ethics committee is also required for any clinical trial of a medicinal product (see Section 2.1.1). Full details of how to conduct a clinical trial of a medicinal product can be found on the MHRA website ( MHRA, 2013 ).

Most human cell-based medicinal products intended for cell therapy or tissue engineering purposes will be classified as ATMPs. If these products are for the EU market then the European Medicines Agency (EMA), Committee for Advanced Therapies (CAT) ( EMA, 2013 ), is responsible for preparing draft opinion on the quality, safety and efficacy of each ATMP for which a marketing authorisation is submitted. If opinion is favourable the MHRA will be responsible, in the UK, for authorising the clinical trial, inspecting the trial and issuing a manufacturing licence.

The UK National Institute for Health Research (NIHR) has produced a very useful online toolkit ( NIHR, 2013 ). This provides practical advice and information on best practice and current legal requirements for conducting clinical trials in the UK. Similarly, the UK Department of Health and MRC have produced a UK Stem Cell Tool Kit ( MRC, 2009 ) as an online regulatory tool for those conducting human stem cell research in the UK.

2.1.6. Ownership and patent rights

There are many who might lay claim to the ownership of specimens and their derivatives, including the donor and relatives, the surgeon and pathologists, the hospital authority where the sample was taken, the scientists engaged in the research, the institution where the research work was performed, the funding body and any collaborating commercial companies. The ultimate control of ownership, intellectual property rights and patent rights will need to be negotiated by the various interested parties. Most universities and research institutes will have a research office that deals with such negotiations, as do most of the larger funding agencies.

2.1.7. Material-transfer agreements

An MTA is a legally binding contract governing the transfer of research materials between two organisations where the recipient intends to use the materials for his or her own research purposes. Biological materials including reagents, cell lines, plasmids and vectors are the most frequently transferred materials and the MTA will define the rights of the provider and recipient with respect to the materials and any derivatives. This should include details of ownership, intellectual property rights and patent rights. The MTA should be signed by the legal representative of both the provider and recipient before any materials are transferred. If primary human tissue or cells are involved the MTA should include a statement confirming that ethical approval and informed consent have been obtained and the recipient should confirm that, on receipt, they will become responsible for using, storing and tracking the material in full compliance with the HT Act. The provider may also wish to state that no liability can be accepted for any problem arising from the use of the cells or tissue and that no guarantee of freedom from microbial contamination can be given.

Restrictions on the use of transferred cell lines should be minimal, but it is reasonable to insist on acknowledgement and even co-authorship where the originating laboratory has made a substantial contribution to the subsequent work. However, merely supplying a cell line would not in itself normally warrant co-authorship of any paper describing work carried out using that cell line. The MTA should also indicate that the cells must not be passed on to a third party or used for commercial exploitation. If the recipient derives a sub-line by cloning and/or genetic manipulation, then a new agreement of ownership will need to be established and this proviso should also be contained in the MTA. Again most universities and research institutes will have a research office that deals with agreeing and issuing MTAs, and will usually arrange for them to be signed by a legal representative.

2.1.8. Creating cell lines from animal tissues

In the UK the use of animals for research purposes is regulated by the Home Office and must comply with the Animals (Scientific Procedures) Act ( Home Office, 2012 ) 1986 as amended in 2012. These amendments transpose European Directive 2010/63/EU ( EU Directives, 2010 ) on the protection of animals used for scientific purposes. Scientists wishing to create cell lines from animal tissues must comply with current legislation. All institutions using animals for scientific procedures will have an Animal Welfare and Ethical Review Body (as defined by European Directive 2010/63/EU) ( EU Directives, 2010 ) and specific approval from this committee may be required if obtaining animal tissues for the creation of cell lines. Further information can be found in the Guidelines for the welfare and use of animals in cancer research ( Workman et al, 2010 ).

2.2. Confirmation of origin

If a new cell line is successfully developed it will be important to confirm the individual and cell type of origin. This will require authentication (see Section 1.2.2) and some degree of characterisation (see Section 1.2.3).

2.3. Preservation

Once a new cell line is established it becomes an important resource. Its authenticity, characteristics and provenance should be recorded (see Section 3.5.1), and cells should be frozen as soon as a sufficient amount is available (see Section 1.4).

3. Cell line practice

Section 3. summary.

Ensure that you are familiar with local Safety Rules as established by the institution’s Biological Safety Officer or advisor.

Handle human tissue samples as potentially infectious material.

Establish correct disposal routes for all types of laboratory waste before starting a procedure.

Ensure that members of staff receive adequate training.

Purchase media and reagents (especially serum) from reputable sources.

Keep media preparation entirely separate from procedures involving living cells.

Record all batch numbers of reagents and media.

Establish ‘standard operating procedures’ (SOP) for all routine laboratory procedures.

Ensure that all items of laboratory equipment (cabinets, incubators, autoclaves, water filtration units, etc.) are properly serviced and are working within prescribed limits.

Inspect the cells under an inverted phase microscope before use. For routine culture, inspect cells daily and consult reference photographs of each cell line at different cell densities. Get to know the cells and how they behave under different conditions.

Freeze new cell lines at the lowest passage possible after clearing quarantine. If they need to be frozen before being cleared they should be treated as if they were contaminated.

Detailed information is available on methodology and GCCP (e.g., Coecke et al, 2005 ; Freshney, 2010 ; Davis, 2011 ).

3.1. Safety

The guidelines on operator safety in cell culture presented here are meant primarily for private and academic research laboratories to be used in conjunction with local and national safety regulations and do not replace rules on safety within individual laboratories, as these vary according to local circumstances. The advice of the local Biological Safety Officer should be sought where there is any doubt about the introduction of new materials or procedures.

Employers are responsible for employee safety under the Health and Safety at Work Regulations ( HSE, 1974 ) by providing information, instruction and training and effective protection against hazard in the workplace. The most relevant component is the Control of Substances Hazardous to Health regulations ( COSHH, 2013 ). These regulations foster safe working practices by establishing that any proposed procedure is both justifiable and safe by requiring that a risk assessment is made before work is started. The COSSH regulations also set out a duty for employees to collaborate fully so that employers can meet the legal obligations. The risk assessment should be approved by the local authorised Biological Safety Officer or advisor ( HSE, 1999 ). It should deal with the entire process and not just individual hazardous chemicals and biological agents. Risk assessments should not be copied from one laboratory to another since the same hazards represent different risks according to local conditions and the scale of the operation.

With regard to product safety, cell culture in the commercial sector is subject to strict regulation. For example, where cell culture products are to be used by the pharmaceutical industry, good manufacturing practice (GMP) ( MHRA, 2007 ) must be complied with, along with the more specific guidance contained in a number of other documents issued by the European Union (EU) ( EMA, 2007 ), the US Food and Drug Administration ( FDA, 2010 ), the World Health Organisation ( WHO, 2013 ) and the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use ( ICH, 2013 ).

3.1.1. Liquid nitrogen

Possibly the greatest hazard encountered in the cell culture laboratory derives from LN 2 , which is used extensively in the freezing and long-term storage of cells and can injure by causing cold burns and frostbite or kill by asphyxiation or by the explosion of poorly designed LN 2 containers. A worker at a laboratory in Edinburgh died from asphyxiation by LN 2 evaporation-induced oxygen depletion in 2000 ( BBC News, 2000 ) and there was a similar case in Australia in 2007 ( Finkel, 2007 ), and a factory in Japan was destroyed by the explosion of a storage vessel in 1992 ( HSE, 1992 ). Thus it is essential that all appropriate safety measures for the handling and storage of LN 2 , as identified in the relevant risk assessment, are in place in all laboratories and that these are rigorously adhered to; LN 2 suppliers are a useful source of information and the latest regulations. Ultimately, however, local factors may be of critical importance (e.g., room locations and sizes, alarm and air-handling systems) and these MUST be factored into the risk assessment. The storage area should be well ventilated and there should be an oxygen deficiency alarm and mechanical ventilation (preferably activated through the oxygen monitor).

A further hazard associated with LN 2 is the risk of explosion where vials are stored submerged in LN 2 . This problem was at its most acute when glass ampoules were widely used, but it still exists with poorly sealed plastic ampoules where LN 2 is drawn into the vial during storage and causes a potentially dangerous explosion when the vial is warmed at thawing. Ideally, vials should not be submerged in LN 2 , but if they are, a protective wrapping such as Cryoflex (Nunc, Thermo Scientific) may be considered.

Appropriate personal protective equipment (e.g., insulated gloves, boots, waterproof apron and face masks) and equipment for safe manual handling of nitrogen vessels should be available. Staff should also receive training in safe working practices for the LN 2 storage facility. Access to storage vessels should be strictly controlled.

3.1.2. Biohazards

The main hazard arising from cell cultures themselves is from infectious agents carried either by the cells or from the components of the culture medium. Cells can carry viruses and at least one fatality due to a viral infection acquired from cells has been reported ( Hummeler et al, 1959 ). Sera could also contain a variety of microorganisms, including viruses and mycoplasma.

The biohazard risks associated with cell culture can be minimised by GCCP and appropriate containment and disposal protocols. Laboratory workers should also use personal protective equipment such as a lab-coat, gown or coveralls. Gloves and suitable eye protection are also recommended, depending on the task and the level of risk.

The use of blood or tissue from laboratory staff for the development of cell lines is not recommended, particularly for the generation of transformed cell lines, as the person concerned would have no immunity to the transformed cells.

3.1.3. Clinical specimens

Testing of the donor cannot be used as proof of absence of infection of the cell line, as contamination may occur by various sources in cell culture, and tests based on a blood sample taken before the cells were donated may not reflect the actual microbiological status of the donated tissue. Comprehensive advice on working with potentially infectious material in the laboratory is contained in guidelines from the Health and Safety Executive ( ACDP, 2005 ). Material with a high potential risk of infection should be excluded or handled appropriately. All samples of blood, body fluids, secretions, tissues and cells are potentially infectious and must be handled at Containment Level 2 in a Class II MSC. Risk of exposure to infection can be minimised by avoiding the use of ‘sharps’ (such as needles and blades) and any items or processes likely to create aerosols. After taking blood, the needle should be removed from the syringe, not resheathed, and discarded safely into a ‘sharps’ container, before the specimen is transferred. You may wish to consider whether vaccination, for example, hepatitis B and tetanus, should be considered for laboratory workers handling human or animal tissue.

3.1.4. Primary cultures and finite cell lines

It is best to treat all cell lines as potential sources of infectious agents and handle accordingly; therefore, the above precautions should be maintained with any cell lines derived from clinical samples. There are documented cases of serious laboratory-acquired infections (e.g., hantavirus, lymphocytic choriomeningitis virus) from tissue, primary cell cultures and tumour cells taken from, or transplanted into, rodents ( Lloyd and Jones, 1984 ). When obtaining primary tissue from laboratory animals it is important to ensure that the animals used are free of specific pathogens (SPF) and suppliers should provide evidence of testing. Alternatively, if the animals have been infected deliberately as part of an experiment, or are otherwise suspected of carrying a specific pathogen, tissues obtained from them must be handled appropriately, including the relevant level of containment. This information should be used in risk assessments and cross-referenced in laboratory record books where the respective primary cells are used. It should be assumed that any hazards associated with primary cultures will also be present in cell lines derived from them.

3.1.5. Stem cell lines

In principle, the infectious hazards that may arise with stem cell lines are no different from any others in that workers should consider the likelihood of contamination with pathogens associated with the species and tissue of origin. In the case of hESCs the risk of contamination of the original donor tissue with the most serious blood-borne pathogens is very low ( Zou et al, 2004 ). However, when stem cell lines are differentiated to form tissue cell types they may provide a suitable culture substrate for the growth of pathogenic viruses such as HCV, HBV ( Si-Tayeb et al, 2012 ) and other pathogens depending on the cell types generated ( Bandi and Akkina, 2008 ). Thus, when planning experiments to provide a particular differentiated cell type, consideration should be given to the most likely contaminants that may arise in reagents, cells and any test samples that might replicate in the differentiated cell types. Human iPSCs can be isolated from a broad range of tissues; therefore, the risk is associated with the tissue.

Human and mouse feeder cell lines used to grow stem cells may also carry viruses and can present similar risks to those for continuous and finite cell lines (see Section 3.1.6). In addition, where primary mouse embryo fibroblasts (MEFs) are used to culture stem cells a range of viruses may occur in the original colony, so a viral screen should be obtained for the MEFs and mycoplasma testing performed (see Section 4.2.4 and Table 3).

3.1.6. Continuous cell lines

The extensive safe use of continuous cell lines indicates that there is little risk from routine cell culture. However, as most cell lines are not fully characterised, they should be subjected to local risk assessment by the local Biological Safety Committee (BSC). A tumour grew in a laboratory worker accidentally inoculated with cells of a human tumour cell line through a needle ( Gugel and Sanders, 1986 ) and cancers have been transferred between people during transplantation ( Stephens et al, 2000 ). Although the growth of tumour cells from a different person is unlikely in healthy individuals, anyone with a compromised immune system is at greater risk.

3.1.7. Genetically modified cells

The introduction of genes can reactivate dormant infectious agents in the host cell or create new agents by recombination. Viral vectors that can infect human cells (e.g., amphotropic retroviruses) are particularly dangerous. Recommended procedures for creation, use, storage, transportation and disposal of genetically modified organisms, including modified cell lines, are given in the Genetically Modified Organisms (GMOs) (Contained Use) Regulations, UK, 2000 and its subsequent amendments ( HSE, 2000 ) ( nb : these do not apply to construction of somatic cell hybrids). These regulations describe how to make a full risk assessment, which must receive approval from the Local Genetic Modification Safety Committee and, in certain cases, specific approval from the HSE may be required.

Genetically modified cells may require special conditions. For example, selective pressure may need to be maintained on transfectants to retain the genetic modification and the pressure may need to be maintained during storage. Distribution of genetically modified cells may be subject to regulation, depending on the modification.

3.1.8. Containment

The HSE guidance on what level of containment is required for working with potentially infectious material, including human tissue samples states: ‘Laboratories that work with potentially infectious material, but where it is unlikely that group 3 or 4 agents are present should achieve Containment Level 2 as minimum’ ( HSE, 2005 ). Long-established continuous cell lines may be handled at Level 1, subject to the approval of the BSC, but in practice it may prove to be more convenient for all tissue culture facilities to be maintained at the same level, that is, Level 2. This level of containment is also applicable to untested cell products such as monoclonal antibody-containing supernates and cell homogenates. These HSE guidelines also recommend that all subculture, or other procedures involving the manipulation of bulk cells, should be performed in a Class II MSC. Laminar flow devices other than MSCs should not be used for cell culture. Horizontal flow cabinets, where the airflow is directed at the operator, are particularly hazardous and must never be used when working with cells that are known to, or may, carry pathogens, or with potentially infectious cell derivatives. Horizontal laminar flow cabinets are still used by laboratories working with early embryos and ES cells that are known to be pathogen-free. However, the use of this type of cabinet should be strictly controlled and subject to local risk assessment and approval by the local safety committee. Modifying MSCs and other contained cell culture hoods, for use with microscopes, may disrupt airflow so much that they are neither safe for the operator nor provide adequate protection for the cell cultures. Such modifications should only be made following approval by the local safety committee and any modified equipment should always be re-validated before use.

The spread of infection often occurs via contaminated aerosols and any process that produces aerosols from crude cell culture preparations is a potential source of infection. Such processes (e.g., centrifugation, tissue disaggregation, vortex mixing and sonication) should be contained or the material rendered harmless before it is processed. There are special guidelines for the safe use of flow cytometers with unfixed cells ( Schmid et al, 2007a , 2007b ).

Any cell samples to be submitted for specialist microscopy services (e.g., confocal microscopy or scanning/transmission electron microscopy), or other external services should first be discussed with the responsible microscopist and Biological Safety Officer or advisor and should be subject to appropriate risk assessment.

Detailed information on cell culture laboratory design, use of MSCs and GCCP is widely available ( Coecke et al, 2005 ; Freshney, 2010 ; Davis, 2011 ).

3.1.9. Disposal

Control of the disposal of laboratory waste should prevent exposure of staff and environment to infectious hazards and prevent contamination. In the UK those producing clinical waste (including drugs, pharmaceuticals, animal and human material and any items contaminated with these materials) have a duty in law to ensure its safe disposal ( Environmental Protection Act, 1990 ). All infected waste arising from work in laboratories should be made safe to handle by appropriate means (e.g., autoclaving), before disposal by incineration. England and Wales, Scotland and Northern Ireland each have their own hazardous waste legislation in the form of statutory instruments and rules to implement the EU Directive on Waste, which sets out a framework within the Member States for controlling the production, transport and disposal of hazardous waste ( Hazardous waste, 2004–2009 ).

3.2. Training

New staff should not be allowed to work in the tissue culture facility until deemed competent. Both practical, hands-on training and theoretical training should reinforce the need to use good aseptic technique and awareness of contamination as an important issue that can be minimised through GCCP. Practical training is best carried out on a one-to-one basis with an experienced member of staff, with extensive reference made to any relevant SOP. As compliance with any demanding technique tends to decrease with time and familiarity, performance should continue to be monitored.

Individuals experienced in cell culture starting in a new laboratory should read the protocols specific to the laboratory, such as routine handling and monitoring of cell cultures (see Sections 3.5 and 4), safety, waste disposal, autoclaving, incubator use/sharing, labelling of cultures and medium storage.

3.3. Culture reagents

It is recommended that reagents and sera are purchased from suppliers who issue certificates of analysis or results of QC testing with each batch of product. Buy in bulk quantities suitable for the level of usage to minimise batch variation and store at the temperature recommended by the manufacturer. Aliquot proteinaceous solutions, such as serum and trypsin, rather than repeatedly freeze and thaw large bottles.

3.3.1. Media production

Most commercial suppliers offer a custom media service for specialised formulations. Basic media formulations are usually offered both as single strength and as 10 × concentrated liquids by suppliers. Although cost savings can be achieved by using 10 × concentrates, discounts are often available on large orders of 1 × medium and therefore many laboratories have adopted its use. If concentrate is used, this is diluted into bottles containing sterile ultra-pure water. Sterile L-glutamine and sodium bicarbonate are then added and finally the pH is adjusted. The advantage of this system is that it is quick and technically undemanding. However, several points should be borne in mind.

Media concentrates have changes made to their basic formulations, mainly to overcome problems of solubility.

Precipitate is often seen on storage. If the concentrate is aliquoted the precipitate can cause variation between bottles.

Suppliers acidify the medium to improve solubility. This in turn requires significant amounts of base to neutralise the medium.

3.3.2. Powdered media

Powdered media produce more stable uniform products with longer shelf lives than concentrates. However, the process does require specialised equipment for filtration and bottling. Note:

The powder should be free flowing and white to off-white in colour, with no sign of dampness.

The medium should be stirred until all the powder is dissolved. The presence of fine particulate matter may require pre-filtration or a change of supplier.

Medium should always be prepared and filtered on the same day.

Sterilisation requires filtration to a pore size of 0.22 μ m. Cellulose filters are most common but polyvinylidene fluoride (PVDF) filters should be used when protein is present in the medium. Although a 0.22- μ m filter will prevent the passage of bacteria and fungi, mycoplasma can pass through pores of >0.1 μ m in diameter; therefore, most commercial suppliers now filter to this limit. However, it is still advisable to screen regularly for mycoplasma contamination rather than assume its absence from media. It is difficult to exclude viral contamination and pre-screening of natural products, such as serum, by the supplier is usually the only option.

Single-use disposable cartridges or filter flasks are recommended as the most convenient option for media filtration. Note:

The equipment should be dedicated for media production only.

A Class II MSC should be dedicated to media and supplement production (horizontal laminar flow may be used provided there are no antibiotics or toxins in the medium). If a dedicated cabinet is not possible, then it should not have been used for cell culture for at least 1 hour. It must also be cleared of all equipment and thoroughly cleaned with 70% alcohol or non-corrosive disinfectant.

All tubing should be clean and autoclaved before use and connections should be securely in place.

Sterile bottles and caps should be stacked outside the cabinet and introduced one at a time to receive medium. Stacking bottles within the flow cabinet will seriously compromise the airflow and consequently sterility.

During bottling, representative samples should be drawn off at regular intervals. These samples should then be incubated at 37 °C for at least 10 days to check for contamination.

Bottled media should be stored at 4 °C in the dark.

Some medium components such as glutamine are heat labile ( Ozturk and Palsson, 1990 ). Glutamine degradation occurs at ∼ 3% per month when stored at 2–8 °C, but the rate of degradation increases if the medium is warmed to 37 °C. This is best addressed by adding glutamine when the bottle is first used and by discarding bottles after a set time period (e.g., 1 month). Stabilised forms of glutamine are available that avoid this problem (e.g., Glutamax, Invitrogen).

If any sample shows contamination in repeated samples, the whole batch of medium should be discarded.

3.3.3. Serum batch testing

Simple preliminary tests can help avoid the disastrous consequences of using media, sera or supplements that do not adequately support cell growth. Many laboratories buy large batches of serum (held in store by the supplier) to avoid batch variability; batches can be kept at −80 °C without noticeable loss of function. Batch testing of serum should use a relevant range of cell lines and may include criteria for (a) cell attachment and spreading, (b) cloning efficiency, (c) growth rates and, where appropriate, (d) a functional assay and/or colony morphology. Low serum concentrations (e.g., 1%) can help highlight differences between sera. It is important to limit carry-over of the old serum during testing, as this could mask differences between the old and new batches. Trypsinisation of adherent cells will usually remove most of the serum, but cells growing in suspension will need to be centrifuged. The selected batch may be held on reserve for a maximum of 1 year and delivered as required.

3.3.4. Defined media and serum replacements

Developing a defined medium, that is, serum-free and preferably free of all impure or undefined products, for a particular cell line or cell type can be very time consuming. There is a wide range of serum-free media available (see Box 3 ), although not all of these are totally defined. Any protein supplementation should be with recombinant protein. The advantages of defined media include standardisation, reproducibility, absence of microbiological contaminants and the potential for selective culture of specific cell types. However, they are generally more expensive and will mean that one medium may not suffice for all cell lines in use. Alternatively, a regular medium such as DMEM or DMEM/F12 may be supplemented with a serum replacement (see Section 6.6). Some of these, for example, SIT (Sigma) are defined (selenium, insulin and transferrin), but others are proprietary mixtures and undefined and should be batch-tested as for serum. Fully defined or at least serum-free medium should be used whenever possible.

3.4. Equipment

3.4.1. microbiological safety cabinets.

Most cell culture is undertaken in a Class II MSC. These cabinets provide protection to the operator as defined in the BS EN 12469:2000 ( British Standards Institute, 2000 ) and protect the external environment while maintaining a clean working environment, but give no protection against toxic, radioactive or corrosive materials for which specialised cabinets are required. The effectiveness of a MSC is dependent on its position, correct use and regular testing.

Cabinets should be sited away from doors, through traffic and air-conditioning inlets. Movement in the area of the MSC will disturb the airflow and therefore access to the area should be restricted to essential personnel. Recommendations for siting MSCs are given in BS5726:2005 ( British Standards Institute, 2005 ).

All MSCs should be tested annually for airflow, operator protection factor and filter integrity. This should be increased to every 6 months where GMOs or primary unscreened human material are used ( HSE, 2001 ). Cabinets used for general cell culture should be tested annually. Testing and servicing should be carried out by trained competent personnel. Before servicing and testing is carried out, adequate fumigation is required. This is usually performed using formaldehyde gas but some manufacturers offer vaporised hydrogen peroxide (VHP) sterilisation, which leaves no toxic residue (see Table 2 ). Advice should be sought from the local safety committee regarding whether this is acceptable in your institute. Training is essential before either procedure is carried out. An equipment safety certificate is normally required by servicing engineers before testing can begin.

When performing cell culture work within an MSC it is important to minimise the potential for contamination of the working environment and cross-contamination between cultures. This can be greatly assisted by the following:

Swab down the inside of the cabinet and the work surface with 70% alcohol before starting.

The inside of an MSC and items that you bring into it should be clean but are not sterile, and good aseptic technique requires that you do not touch any of the surfaces with sterile instruments, pipettes, and so on.

Do not make rapid movements within the cabinet, as this may disrupt the airflow.

Manipulate fluids slowly and gently with the assistance of a pipetting aid to avoid the creation of aerosols.

Never have more than one cell line at a time in the cabinet.

Do not overcrowd the cabinet and never obstruct the grills or front opening.

Organise the work area such that sterile reagents and cultures do not come in contact with each other.

Use caution when homogenising tissues or cells in an MSC. If high-energy processes such as sonication are used the particles cannot always be assumed to be contained by the cabinet airflow.

Clean and decontaminate the cabinet inner surfaces after each work session and periodically decontaminate the tray under the MSC working surface using 70% alcohol or a non-corrosive disinfectant.

Some MSCs have an ultraviolet lamp installed to assist with disinfection of the cabinet. Although ultraviolet light can be useful, its effectiveness is limited and it should not replace other decontamination procedures.

A Bunsen or similar burner must not be used when working in a MSC, (unless absolutely required for a specialised procedure) as they disrupt the airflow pattern, reducing the cabinet's effectiveness, and they pose a fire risk.

3.4.2. Incubators

Incubators are used during cell culture to maintain an optimal cell growth environment by controlling the temperature, humidity and carbon dioxide concentration. Most modern incubators are humidified and used with an atmosphere typically containing 5% CO 2 , although other concentrations may be required depending on the bicarbonate concentration of the medium. The following points should be considered:

Incubators should be chosen carefully with reference to their expected use and any desirable features that can be included within budget (see Box 4).

Humidifying water can contain an antibacterial/antifungal agent or other appropriate contamination control measures (see Box 4), but only if checked beforehand for any possible toxic effects on the cultures (e.g., by performing a plating efficiency assay).

Incubators should be calibrated for temperature and gas composition.

CO 2 levels should be checked monthly using a calibrated CO 2 meter (marked deviations will be evident as a change in pH of the medium).

Every 6–8 weeks the incubator should be emptied, dried and cleaned with 70% alcohol or equivalent non-corrosive disinfectant. All shelves should be similarly removed and cleaned.

Individual trays on which culture flasks can be easily moved in and out of the incubator should be used to reduce contamination from spillages.

Incubator temperatures and contents should be inspected daily.

Spillages must be dealt with immediately.

All infected plates, dishes or flasks must be removed immediately and disposed of appropriately.

Incubators must only be used for cell culture and not for incubating microorganisms or biochemical samples.

Gassed incubators should be attached to a suitable cylinder change over unit or protected central supply.

Cylinders used to supply gas should be securely anchored.

Cylinders should be clearly labelled and have the correct regulating valves attached. The tubing should be appropriate for the pressure of the gas used and securely fastened to avoid any leakage of carbon dioxide, which is a potential asphyxiant.

A 0.22- μ m porosity, non-wettable filter should be used on the input gas lines.

Cylinders should be changed by trained personnel wearing suitable high-impact eye and foot protection.

It is preferable to have the CO 2 supply cylinders or tank located outside the sterile area to minimise disturbance when changing or refilling.

3.4.3. Hypoxic incubators

Although atmospheric oxygen concentrations and those inside standard 5% CO 2 cell culture incubators are close to 21%, in vivo concentrations are much lower, ranging from 1–12%. The use of hypoxic incubators and chambers is becoming increasingly widespread in order to mimic in vivo conditions using in vitro cell culture models. In particular hypoxia-inducible factors have been shown to influence tumour cell metabolism, angiogenesis, growth and metastasis ( Bertout et al, 2008 ). Hypoxic conditions have also been suggested to be important in maintaining pluripotency in hESC cultures ( Lengner et al, 2010 ). Hypoxic cell culture is often performed using ‘tri-gas’ incubators where a nitrogen supply is used to displace and reduce oxygen levels within the incubator, typically to a range between 0.5 to 2%. This type of incubator has the disadvantage that hypoxic conditions will be lost when the door is opened or when cell cultures are removed. To maintain stringent hypoxic conditions it will be necessary to use a specialist hypoxic chamber/workstation. These are sealed units that maintain the required hypoxic atmosphere and typically have sealed glove ports that allow cultures to be manipulated without removing them from the unit. They usually also have an air lock to allow media, reagents and consumables to be transferred into the unit without losing the hypoxic environment.

3.4.4. Microscopes

Most tissue culture rooms are equipped with an inverted light microscope that allows the user to inspect their cell cultures regularly and to perform cell counts (see Section 3.4.6). A suitable range of objective lenses would be × 4, × 10 and × 40 with × 10 eyepieces. Image contrast is enhanced using phase contrast optics. It is preferable to have a trinocular head to accommodate a digital camera. Cell images can be captured and stored to monitor any morphological changes that take place in the culture, an essential element of QC. Fluorescence microscopes are also commonly used in tissue culture labs where fluorescent labels have been introduced into the cells or the animals from which cell cultures are derived. These may require all or part of the lab to be light-free.

3.4.5. Live-cell imaging

Live-cell imaging can be defined as the study of living cells using images that have been acquired from microscopes or screening systems. It can be used for cell line QC and for the study of cellular dynamics. Real-time cell-imaging systems allow the user to monitor live-cell growth and any changes in morphology that could occur as a consequence of differences in cell density or culture conditions without intervention. They are less labour-intensive and can save money on plastics and media. However, the user must be aware of which parameter is being used to measure growth as this will influence kinetic analysis if growth rates are being calculated. Real-time cell-imaging systems have been developed by many companies and some examples include Incucyte (Essen), xCELLigence (Cambridge Bioscience), CellVivo (Olympus), Cell Observer (Zeiss), Cytation3 (BioTek Instruments), DeltaVision Elite (GE Healthcare), CloneSelect Imager (Molecular Devices) among others.

The advent of high-content analysis (HCA), in which cell images that have been captured using a high-resolution light microscope are extracted and quantitative data is analysed in an automated process, is having a great impact on research as multiple markers at subcellular resolution can be measured in large-scale quantitative assays. Examples of high-content systems include the Opera and Operetta (PerkinElmer), CellInsight NXT HCS Platform (Thermo Scientific), Cell-IQ (Chipman) and the multi-plate imaging system Biostation CT (Nikon).

3.4.6. Cell counting

Cell counting, manual or electronic, is essential to determine growth rates accurately and to set up reproducible experiments. Manual counting is usually performed using a hemocytometer (Improved Neubauer), which consists of an engraved glass microscope slide and thick coverslip creating a chamber where the cells are placed and then counted under a microscope. As the volume within the chamber is known the cell count per unit volume can be calculated. The hemocytometer is cheap and allows visual inspection of the cells, but cell counting is labour-intensive, particularly if many cell lines are used, and variable between users. Electronic counters are expensive but the reproducibility and speed that they provide are of importance when dealing with many cell lines, although they are prone to error, particularly if the cells are clumped.

3.4.7. Autoclaves and sterilising ovens

Autoclaves are used for sterilising equipment and consumables. Safe operation is described in the HSE guidelines ( HSE, 2012 ) and some advice on correct function has been provided by the Centers for Disease Control ( CDC, 2008 ). Autoclaves must be covered by insurance, which will necessitate an annual inspection. It is essential that proper protective clothing (including a face visor and heat-proof gloves) are used and the autoclave not opened until the temperature has fallen below 50 °C. Autoclaving of liquids in glass containers can present particular hazards: autoclave bottles with the caps slack and set a limit on the volume autoclaved to give consistent sterilisation and to leave a standard volume after autoclaving. Leave autoclaved liquids to cool before moving off the autoclave tray or trolley, to reduce the risk of boilover from superheated liquids.

It is essential that regular checks are made to ensure that the autoclave is operating at the required temperature and pressure. Qualitative indicators (e.g., autoclave tape) are useful to distinguish items that have been autoclaved from those that have not, but they do not guarantee that the item is satisfactorily sterilised. Some indicators (e.g., Thermalog, Bennett Scientific) confirm that the minimum requirements of heat and humidity have been met for full sterilisation but must be located at the centre of the load (inside a replicate vessel if necessary). Autoclaving of a test sample (e.g., spore strip testing) may be necessary to confirm sterilisation when setting up a procedure for the first time.

3.4.8. Water-purifying apparatus

The use of ultra-pure water is essential for successful cell culture. Reverse osmosis followed by passage through mixed-bed ion-exchange resins and carbon and micropore filtration provides pyrogen-free water of tissue-culture grade. Water should be measured for pH, conductance and total organic carbon (TOC) ( Whitehead, 2007 ). Serum can protect cells from toxins and consequently the use of ultra-pure water is critical in low protein or serum-free conditions. The purity of water is only maintained if it is placed in suitably clean bottles dedicated to storage of water or media.

3.5 Quality control, record keeping and research integrity

3.5.1. records.

Details of all routine and experimental procedures should be recorded as they are generated. Good practice requires that records be dated, legible, clear in content and made in blue or black ink directly into a bound laboratory notebook or onto a standard form. Each piece of work must have an aim/objective, method, equipment used, results and conclusion and must be signed at the end of the record. Enough detail must be recorded to enable the work to be reproduced exactly. Standard operating procedures (SOPs; see below) should be referenced wherever possible. More stringent requirements may apply to work done under GLP, GMP or GCP(L) conditions.

Records of routine procedures carried out, such as cell counts, cell-line passaging and medium preparation can be kept on standard forms designed for the purpose. These should be stored in a dedicated file, which may be electronic and cross-referenced in experimental notebooks as required. The advent of tablet computers means that primary records may be electronic and linked to a central database, but some laboratories may insist on hard-copy lab books with numbered pages for primary records. If electronic notebooks are used, care should be taken to protect and back up records and to allow ongoing access regardless of changes to the software or hardware used. Procedures should be in place to validate the authorship, date and content of entries; this is particularly important if a patent or other commercial application may arise from experimental work.

The originals of all experimental records remain the property of the funding agency or laboratory and must be lodged with them when an experimenter leaves that laboratory or changes funding agencies. Such records should be securely archived, with systems in place to permit easy retrieval along with protection against tampering.

3.5.2. Quality control

A Certificate of Analysis should be requested from the supplier for each batch of material and this should be stored securely for future reference with the date received in the lab book or dedicated file.

Quality assurance of cultured cell lines, in terms of their authentication, stability and contamination are dealt with above (see Section 1).

3.5.3. Standard operating procedures

Procedures that are regularly performed in a standard manner are best documented in the form of an SOP. This is a clear and detailed list of instructions, written such that suitably trained individuals can understand and perform the task in the intended manner. It should include details of the equipment, reagents and techniques to be used, as well as methods for calculating and interpreting the results. Ideally, each laboratory or organisation should have its own system for the issue, tracking and review of SOPs. This should ensure that all copies can be tracked so that all scientists have the most recent versions and that they are reviewed on a regular basis (at least once every 2 years is suggested). All SOPs should be controlled by either a version number or a date. A nominated scientist should be responsible for the issue of all SOPs, such that only one approved version is current, reflecting best procedure and all other versions are removed from use. Ideally, the system should include provision for permanent archiving of all versions and revisions of all SOPs. Where the experimental nature of the work precludes strict adherence to the SOP it is important that the appropriate SOP is referenced and the deviations noted.

4. Cell line problems: identification and elimination

Section 4. summary.

Misidentification remains a major problem, so all cell lines should be obtained from a reliable source and shown to be authentic.

Contamination with mycoplasma is still prevalent and requires good tissue culture practice and frequent testing to ensure that cell lines are clear of contamination.

Other contaminations can largely be avoided by GCCP.

Antibiotics should not be used routinely.

Decontamination should not be attempted unless the cell line is critical and irreplaceable and even then should only be attempted by an experienced laboratory.

Cell lines, particularly continuous cell lines, are prone to genotypic and phenotypic instability requiring regular characterisation and replacement from cryopreserved stocks.

Cultures should always be examined under an inverted phase microscope before any manipulations are performed and frequent assessments should be made of the viability and appearance of the cell population with reference to photographic records.

4.1. Cell line misidentification

One of the most serious and persistent problems is cell line misidentification often resulting from cross-contamination. This means that authentication is required on receipt, before storage and distribution, and after completion of a project (see Sections 1.2.2 and 1.5.1).

4.2. Mycoplasma contamination

Contamination of cell cultures with mycoplasma was first noted in the 1950s but is still regrettably often disregarded. The following important points should be noted:

Mycoplasma contamination is very frequent, worldwide.

Using mycoplasma-contaminated cells can result in erroneous, misleading or false experimental results.

Owing to lack of visible signs mycoplasma-positive cell cultures can go unnoticed.

Be aware of potential sources of mycoplasma contamination (see Section 4.2.2).

Use good aseptic technique and laboratory practices to avoid mycoplasma contamination.

Have an effective quarantine procedure for all untested cell lines.

Establish a regular and continuous mycoplasma-testing programme.

Scientific journals are starting to ask for evidence of mycoplasma testing before accepting papers for publication.

Mycoplasmas and the related Acholeplasmas (collectively referred to as ‘mollicutes’) are the smallest and simplest self-replicating bacteria and are significant in that they have become probably the most prevalent and serious microbial contaminant of cell culture systems used in research and industry today. Owing to the absence of any visible morphological changes or other symptoms mycoplasma infection of cell cultures often goes undetected. However, it is the invisible effects of the contamination on the infected cells that makes it such a serious problem. It is therefore essential that routine mycoplasma testing is performed regularly on all research cell lines to ensure the validity of study results before publication. Although >20 different species of mycoplasma have been isolated from cell cultures, >95% of infections are caused by six prevalent species, which are the following: M. arginini , M. fermentans , M. hominis , M.hyorhinis , M. orale and Acholeplasma laidlawii .

Although primary cell cultures and early passages are less frequently contaminated with reported incidences of between 1 and 5%; continuous cell lines have much higher incidences of between 15 to 35% ( Drexler and Uphoff, 2002 ).

Mycoplasma are unaffected by many of the antibiotics commonly used in cell culture, such as penicillin and can grow to extremely high titres (typically 1 × 10 7 to 1 × 10 8 organisms per ml) in mammalian cell cultures without producing any turbidity in the medium, or other obvious symptoms. In addition mycoplasma are extremely small (0.15–0.3 μ m) and pleomorphic, and will pass through standard 0.22- μ m bacteriological filters (0.1- μ m filters are required for sterilisation). The only assured way of detecting mycoplasma contamination is regular testing.

4.2.1. Effects

The effects of mycoplasma contamination on the host eukaryotic cell are quite variable but have been shown to alter many host cell functions including growth, morphology, metabolism, the genome and antigenicity ( Drexler and Uphoff, 2002 ). Using mycoplasma-contaminated cultures in experiments will therefore clearly call into question the validity and significance of any research data generated and could result in the publication of erroneous experimental results. Research journals are now starting to ask for evidence that mycoplasma-free cell cultures are used in studies before accepting papers for publication. In addition the time and cost involved in cleaning contaminated laboratories, obtaining new cell cultures and repeating experiments is significant as is the potential reputational damage of publishing erroneous results.

4.2.2. Sources

Common sources of mycoplasma contamination in the laboratory include:

Cross-contamination from other mycoplasma-positive cell cultures.

Laboratory equipment and work surfaces.

Laboratory personnel (often via respiratory tract infections).

Cell culture media, sera and reagents.

The liquid phase of LN 2 cryostorage vessels.

Feeder cell cultures.

Laboratory animals

4.2.3. Prevention

The first step in avoiding mycoplasma contamination is being aware of the most common sources of infection, as outlined in the previous section and then to adopt working practices that reduce the risk of contamination from these sources. First and foremost is the importance of adopting proper aseptic cell culture technique ( Coecke et al, 2005 ; Freshney, 2010 ). Failure to do so will result in equipment, work surfaces, media and reagents rapidly becoming contaminated and leading to the spread of infection. Routine regular cleaning of all MSCs and incubators, including the use of sterile water for humidification of incubators, will help minimise the risk of mycoplasma contamination.

Working with only one cell culture at a time and using dedicated, separate, media and reagents for each individual cell line will greatly reduce the risk of cross-contamination and spread of infection.

Having an effective quarantine procedure in place will also minimise the risk of introducing mycoplasma contamination into the laboratory. All cell lines of unknown mycoplasma status, in particular cell lines brought in from external laboratories and collaborators should initially be quarantined (see Section 1.2.1) until tested negative for mycoplasma. Only then should the cell cultures be transferred to the clean cell culture laboratory.

The overuse and reliance on antibiotics rather than good aseptic technique can result in higher mycoplasma contamination rates and mycoplasma are typically partially or completely resistant to the antibiotics commonly used in cell cultures such as penicillin and streptomycin.

4.2.4. Detection

There are four basic steps to any successful and reliable mycoplasma testing and detection programme.

Test all actively growing cell lines at regular intervals.

Only cell lines previously tested and confirmed as mycoplasma negative should be used in your clean cell culture lab. All non-tested cell lines must be quarantined until they are tested negative for mycoplasma.

Test all continuously maintained cell cultures at defined regular intervals (typically monthly to quarterly, depending on individual risk assessment). Maintain these cultures, where possible, on a short-term basis (2–3 months only) before discarding them and replacing with fresh vials from the same tested working stocks. This strategy not only reduces the amount of testing required but also reduces the problem of culture evolution and genetic drift.

Make sure all cell lines are mycoplasma tested before use in a clean cell culture room.

A large number of simple, reliable, sensitive and specific tests are now available to detect mycoplasma contamination in cell culture (see Table 3 ). When choosing a test method or a test kit, consider the sensitivity of the assay (usually recorded as colony-forming units per ml, or ng ml −1 for PCR-based kits). The number of species detected is also important. Positive control samples may be kept where the appropriate microbiological expertise and proper quarantine facilities are available; otherwise it is better to avoid keeping infected material. For those laboratories not able or not wishing to perform their own mycoplasma testing there are commercial companies and organisations that offer a comprehensive mycoplasma-testing service including Mycoplasma Experience Ltd. ( www.mycoplasma-exp.com ) and the HPA Culture Collections – ECACC ( www.phe-culturecollections.org.uk ).

4.2.5. Eradication

The first step is to autoclave, or disinfect, the contaminated culture and associated media and dispose of them. Next thoroughly clean and disinfect ( Table 4 ) all hoods, incubators, centrifuges, refrigerators, microscope stages and any other equipment, including pipettors, that may have been in contact with the contaminated cultures. It is recommended that potentially contaminated MSCs are sterilised using a suitable chemical fumigant. Using a liquid disinfectant alone may not be sufficient as it is difficult to reach all internal surfaces of an MSC with disinfectant. All media, media components and other reagents used for the contaminated cell lines must be discarded and all other cell lines in use within the laboratory should be quarantined and tested for mycoplasma to detect any spread of contamination.

4.3. Contamination by other microorganisms

With correct working practice it should not be necessary to use antibiotics to control contamination in established cell lines and their use should be discouraged. Microbial contamination may be obvious, indicating that the culture should be discarded, but, if antibiotics are used, contamination may be repressed but not eliminated. Such cryptic contamination may co-exist with the cell culture and only appear when the culture conditions change or the organism develops antibiotic resistance. In addition as antibiotics and antifungal agents act by inhibiting biochemical functions of the organism, these activities may also affect animal cells prejudicing the outcome of experiments. For example, amphotericin B is a membrane active agent and may therefore interfere with any mammalian cell experiments involving membrane trafficking or intercellular signalling.

4.3.1. Bacteria and fungi

If cells are cultured in antibiotic-free media as recommended, contamination by bacteria, yeast or fungi can usually be detected by an increase in turbidity of the medium and/or a change in pH (typically acidic with many bacteria giving a yellow colour change in media containing phenol red as a pH indicator but can be alkaline, pink, with some fungi). It is recommended that cells are inspected daily and must always be examined under an inverted phase microscope before use in an experiment.

The two methods generally used for bacterial and fungal detection are microbiological culture in special media and direct observation using Gram’s stain, although bacterial infections may also be revealed during routine testing for mycoplasma by Hoechst 33 258 staining. If necessary, the help of a hospital microbiology laboratory may be sought with identification and antibiotic sensitivity testing.

If a cell culture is contaminated with bacteria or fungi, then the best method of elimination is to discard the culture and initiate fresh cultures from frozen stock. In the case of irreplaceable stocks, it may be necessary to use antibiotics; the more antibiotics that are tested, the greater the chance of finding one that eliminates the infection. However, if the cells have been routinely grown in media supplemented with antibiotics (which is not recommended), it is almost certain that the contamination will be with organisms that are already resistant to this and some other antibiotics.

To eliminate infection, the cells should be cultured in quarantine (see Section 1.2.1) in the presence of the antibiotic for at least three passages. If the contamination appears to be eradicated, a sample of the culture should be cryopreserved and the remaining cells cultured in antibiotic-free medium for 1 month before re-testing. If clear, the preserved stock can then be used to generate an archive frozen stock for future work; if not, a different antibiotic can be used. It is important to remember that the more antibiotics that are used and fail the more dangerous the organism becomes as a potential contaminant of other cultures.

4.3.2. Viruses

As long as cell culture reagents of biological origin are used, such as serum to supplement media and natural trypsin for subculture, there will always be a risk that endogenous infections in the source of the reagent will infect the culture. Any viral contaminant that grows in the cells will affect the cells’ metabolism and could also present a safety hazard to lab workers. The source of viral contamination can be from the tissue from which the cells are derived (e.g., HIV from Kaposi’s sarcoma cells, EBV from lymphoma cells). Alternatively, contamination can be derived from other infected cultures or, as a more remote possibility, from laboratory personnel. Another route of infection can be during passage of cells in experimental animals, important when considering the use of cell lines for or from implantation of xenograft tumours. Not only do the cells to be implanted need to be free from contamination by extraneous viruses but also the animals into which the transplant is to be made should not harbour viruses that could affect the growth and response to therapy of the cells under study.

Even more than with mycoplasma, elimination of viral contamination is difficult and is likely to be impossible. However, what is worse, there are no simple universal diagnostic tests to identify viral contamination. Next-generation sequencing techniques potentially offer such screening but are yet to be qualified for routine safety testing. Identifying viruses currently necessitates screening with a wide panel of immunological or molecular probes and may be best done by a specialist testing service. As yet, such testing is largely restricted to human pathogens such as EBV, HIV, HTLV I/II and Hepatitis B & C, and few laboratories screen for animal viruses on a routine basis, although some commercial suppliers and veterinary laboratories do. Use of serum-free medium and recombinant trypsin should help to minimise viral infection from reagents and GCCP will minimise the risk of transmission from one culture to another or to the person handling that culture.

4.3.3. Prions

Transmissible spongiform encephalopathy (TSE), including what is known as bovine spongiform encephalopathy, BSE, or mad cow disease, is unlikely to be present in cancer cells or tissue culture products. Risks of prion contamination may need to be considered when using cell lines from the CNS or from patients with certain diseases associated with abnormal prion expansion. It should be noted that prions are not destroyed by autoclaving or by most chemical disinfectants. Disposal into 10% hypochlorite followed by incineration is recommended for any contaminated material.

4.4. Genetic instability and phenotypic drift

Two other major problems that can affect the utility of cell lines are genetic instability and phenotypic drift, both of which may progress the longer the cell line is cultured. Records should be kept of the length of time a cell line has been kept in culture. For finite cell lines, this is determined by the generation number, the number of doublings since isolation (necessarily approximate as it is difficult to measure the number of doublings in the primary culture). This number will determine the lifespan of the culture as most finite cell lines will die out due to senescence at between 20 and 60 doublings, which means that they can only be used reproducibly between 15 and 45 generations depending on the cell type. Cells are frozen at the lowest generation number possible and used to replace stocks at regular intervals before the onset of senescence. When thawed the generation number resumes at one over the number at freezing. For continuous cell lines, the number of passages since last thawed from the freezer is recorded. If the passage level at freezing is known, this may be added on but often this is not known.

4.4.1. Genetic instability

The chromosomal content of most continuous cell lines is both aneuploid (abnormal chromosome content) and heteroploid (variable chromosome content within the population). Many cancer cell lines have defects in p53 and other genes that monitor and repair DNA damage, resulting in an increased mutation frequency. Hence, the genotype of continuous cell lines can change with time and cell lines should not therefore be maintained for extended periods of time in continuous culture ( Wenger et al, 2004 ; Saito et al, 2011 ).

In general, cell lines derived from normal tissue tend to have a chromosomal content typical of the karyotype for the species of origin. Most normal human cell lines will senesce and cease proliferation without major heritable changes in the genotype. In contrast, rodent cell lines, particularly mouse lines, become unstable and immortalise readily. It is important to check for any critical subculture or other handling measures for any new cell line. For example, immortal cell lines, such as 3T3, can retain their established growth characteristics provided that the recommended maintenance procedures are adhered to. In particular, they should not be allowed to become confluent, but should be subcultured from mid-log phase, and replaced regularly from frozen stock. They require constant monitoring to ensure that transformed variants, readily detected morphologically by their more refractile appearance and lack of contact inhibition, do not overgrow.

4.4.2. Phenotypic instability

Lack of expression of the differentiated properties of the cells of origin is a major recurrent problem. This can be due to selection of the wrong cell lineage in inappropriate culture conditions. For example, a disaggregated skin biopsy will ultimately give rise to a fibroblastic population that overgrows the epidermal keratinocytes, unless selective conditions are used. However, even under selective conditions, the need for propagation stimulates cell proliferation rather than differentiation. This process can either select undifferentiated cells or can lead to a loss of differentiated characteristics. In some cases, such as fibroblasts or endothelial cells, this is due to dedifferentiation, but in others, such as mammary epithelium, it is probably due to propagation and expansion of the progenitor cell compartment, which lacks the differentiated characteristics.

4.4.3. Stability of stem cell lines

There are particular problems associated with cell lines derived from stem cells, whether embryonic, fetal, adult or iPSCs:

Phenotypic stability is dependent on the medium, particularly on cytokines and the activity of specific signalling pathways. For example, most mouse embryonic stem cell cultures will tend to differentiate spontaneously unless the stem cell phenotype is maintained with LIF or a feeder layer that produces it.

If allowed to differentiate, the resultant phenotype is controlled by regulatory factors in the medium, such as retinoic acid or tetradecanoylphorbol acetate (TPA), and the microenvironment, e.g., by the cell density, extracellular matrix and signalling between cell types. Therefore, the plasticity of the phenotype, while not insignificant in other cell lines, is of particular importance in culturing stem cells, such as ESC and iPSC, and any other progenitor-type cell line that is induced to differentiate for experimental use (e.g., HL60, NB4 and SH-SY5Y).

It is not entirely clear whether mesenchymal stem cells are inherently prone to genetic instability and transformation or are made so by genetic manipulation. Some reports claim transformation does not occur ( Bernardo et al, 2007 ; Choumerianou et al, 2008 ), whereas others claimed that it does ( Ren et al, 2011 , 2012 ), although this can be due to cross-contamination ( Torsvik et al, 2010 , 2012 ). Mesenchymal stem cells (often now termed mesenchymal stromal cells) usually do not have a clonal origin and this heterogeneity may explain differences in results. Pluripotent stem cell lines (i.e., human ESCs and human iPSCs), which are usually clonal in origin, are well known to be susceptible to developing chromosomal changes and need to be periodically checked for genetic integrity. Again, it is rare cells in the population that acquire a growth advantage, often at the expense of ability to differentiate, that take over the culture. Culture conditions that are suboptimal are especially prone to this. Re-cloning the cells and screening for sublines with normal karyotypes can work, otherwise reverting to an earlier passage is recommended.

As for any other cell lines, authentication of stem cell lines is essential, and genotypic and phenotypic instability must be assessed.

Examination of processes that depend on the expression of the in vivo phenotype, whether normal or neoplastic, may require modifications to culture conditions (e.g., high cell density, growth factors, low serum, position at the air–liquid interface, heterologous cell interaction and extracellular matrix), which usually are incompatible with cell proliferation. Hence, different conditions need to be defined for culture of a cell line dependent on whether cell proliferation or cell differentiation is required. It is important and probably essential for comparative purposes that different laboratories using the same cell line should match their culture conditions as closely as possible.

5. Troubleshooting

Section 5. summary.

Approach problem-solving in a systematic manner.

Start by looking at what changes have been made: equipment, reagents and media.

Ensure proper records are made of the nature of the problem and how it was solved.

Keep everyone using the facility or equipment informed of the problem and its solution.

Even with full attention to these Guidelines and/or other established rules of good practice ( Coecke et al, 2005 ; Freshney, 2010 ; Davis, 2011 ), every laboratory will, from time to time, encounter problems ranging from widespread fungal contamination to quite subtle deviations from normal patterns of cell growth. When such problems occur, a logical and systematic approach should be taken to identifying and removing the causes.

Without good background information, assessment of a problem can be unnecessarily difficult or impossible. Careful logging of reagent batch numbers used to make each bottle of medium may often seem pedantic and time-consuming, but can prove invaluable when problems occur. Similarly, careful documentation of the normal behaviour of a cell line provides essential background information. This can include records of cell counts at subculture and occasional photographs of growing cultures ideally at both low- and high cell population density.

The following general approach to troubleshooting may prove useful:

Once the existence of a problem is suspected, it is important to define its characteristics and inform all those who may be affected.

If the nature of the problem is readily identified (e.g., a defective incubator), make sure by appropriate means that its existence is known (e.g., a large notice on the incubator) and that the person responsible is dealing with the problem.

Less obvious problems will need a more comprehensive survey of the facts. This may be facilitated by a meeting of all those involved, as even apparently quite trivial observations may be relevant.

Once the problem is identified, it should be possible to draw up a list of possible causes in order of probability.

It is often useful to ask ‘what is new?’ in terms of reagents, including plasticware, equipment or procedures, or even new staff, which may coincide with the problem. Be aware, however, of possible time displacements such as the effects of a minimally substandard medium batch only becoming manifest after several cell generations, with some cell lines being more sensitive than others.

When switching to a new batch of any medium component (including serum), even though this has been batch-tested, retain a reasonable amount of the old batch for some period of time. This will allow head-to-head testing should problems arise when the new batch is introduced.

With problems of deficient cell growth and/or unusual appearance, the problem may lie with the cells, the growth medium, the growth environment or some combination of these. Clues as to which of these to pursue first may come from which cell lines are affected. Do they share an incubator? Do they have a common medium? Are they using the same culture vessels? Have preparation or culture procedures changed?

If a particular cell line is affected, and tests for contamination (bacteria, mycoplasma) are negative, a vial of the cell line should be taken from frozen stock and the old and new cells tested head-to-head over several passages. If the old cells continue to do badly and the new cells grow normally, then the old cell stock should be discarded and the new stock used for future work. If both stocks do badly then the problem probably lies elsewhere (virtually all cell lines take a period of time to recover from being frozen, so this needs to be taken account of when comparing growth patterns). In addition, a genotyping test such as STR profiling should be performed to rule out a possible misidentification. Sterility testing (inoculation of medium from the culture into broth or agar at normal culture temperature and also at 20–25 °C, in order to isolate microorganisms that grow best at lower temperatures) is worth considering to rule out contamination with a slow-growing microorganism.

Where a problem with the medium appears to be present, a series of tests should be set up in which head-to-head growth comparisons are made in different media where only one medium component at a time is changed. Basal medium can also be obtained from an alternative supplier and compared. Although serum and basal medium may be the most obvious sources of problems, other components including water, glutamine and antibiotics are also candidates.

If a problem component is identified, the finding should be discussed with the supplier who may be able to state that the batch has been used by many other laboratories without problems or (occasionally and off-the-record) that there may be a problem with a batch.

Where problems of contamination are encountered, again the first step is to look for any changes in procedures, sources of materials and staff. The records from sterilisation equipment should be checked, and the medium and its components incubated to see whether contamination appears. Determine whether the problem is confined to one person, cell line, incubator or MSC, or is more general. General problems tend to implicate equipment or ventilation failure while specific problems may relate more to a particular specialised reagent or procedure or even to technical lapses of one individual.

In the experience of many workers, growth problems sometimes are never satisfactorily solved, but the cells begin, after some time, to resume normal growth. Such problems may, however, recur and the combined experience of the first and second episodes may be helpful in further investigation. Similarly, problems of contamination often go away without a specific cause ever being identified. A crowded lab will be particularly susceptible and will need strict enforcement of the rules of aseptic technique.

Box 2 Patient consent form: points to consider

Patient consent should only be taken by suitably qualified individuals with the required specialist training and researchers (other than those with medical qualifications) should not typically have any direct contact with donors.

The Patient Consent Form and associated Patient Information Sheet (necessary for most studies) should be written in concise and explicit language that anyone can easily understand, explaining clearly the need for the specimen, the overall objective of the research and why it is important (in lay terms).

The additional discomfort or inconvenience that will occur if the donor agrees to the request should be clearly explained.

The donor should be told clearly that there is no obligation whatsoever to participate in the research.

If the research may be exploited commercially, the donor should be told clearly what financial benefit might be gained from the research and a waiver to commercial rights should be requested.

The donor should be told that the research has been approved by the local Ethics Committee (give date and reference).

All forms should be marked Confidential.

It should be made clear that confidentiality will be assured, but if not (e.g., familial studies) indicate who will have access to the clinical data and how access will be controlled.

Fully informed consent means that the person should have access to all information relating to the use of the specimen provided. The details may be covered in a Patient Information Sheet.

The information sheet and consent form must be printed on official-headed notepaper.

Consent forms should address the following questions:

Have you read the information sheet about this study?

Have you had an opportunity to ask questions and discuss the study?

Have you received satisfactory answers to all your questions?

Have you received enough information about this study?

Which doctor have you spoken to about this study?

Do you understand that you are free to withdraw from this study (i) at any time, (ii) without giving a reason and (iii) without affecting your future medical care?

Box 3 Suppliers of serum-free media or serum substitutes

It should be noted that many of these formulations will contain protein supplements, such as bovine pituitary extract (BPE), which are undefined. Defined supplements that are available include selenium, recombinant insulin (Sigma, Invitrogen) and recombinant transferrin (Merck Millipore: www.millipore.com ) as well as many peptide growth factors (Abbiotec: www.abbiotec.com, and others) and albumin (Novozymes-Biopharma: www.biopharma.novozymes.com ).

Serum-free media

AthenaES: www.athenaes.com/

Atlanta Biologicals: http://atlantabio.com

BD Biosciences: www.bdbiosciences.com/

Cell Applications: www.cellapplications.com

CellGenix: www.cellgenix.com

CellGro: www.cellgro.com

Clonagen: www.clonagen.com

CoaChrom: www.coachrom.com/

Hyclone (GE Life Sciences): https://promo.gelifesciences.com/gi/hyclone

Hycor: www.hycorbiomedical.com

Invitrogen: www.invitrogen.com

Irvine: www.irvinesci.com

Lonza (Clonetics, BioWhittaker): www.lonza.com

Mediatech: www.cellgro.com

Metachem: www.metachem.co.uk

Millipore (Merck Millipore): www.millipore.com/

MP Biomedicals: www.mpbio.com/

PeproTech: www.peprotech.com

Perkin Elmer: www.PerkinElmer.co.uk

PromoCell: www.promocell.com/

Roche Applied Science: www.roche-applied-science.com/

Sigma (JRH Biosciences): www.sigmaaldrich.com

Stem Cell Technologies: www.stemcell.com

Stratech: www.stratech.co.uk

TCS CellWorks: www.tcscellworkscatalogue.co.uk/

Zen Biologicals: www.zen-bio.com

Serum substitutes

Bayer: www.bayer.com

Celox: www.celoxmedical.com/

Lonza: www.lonza.com/

Protide: www.protidepharma.com/

Roche Applied science: www.roche-applied-science.com/

Sigma: www.sigmaaldrich.com

Web addresses last accessed July 2014.

Box 4 CO 2 incubators

Choosing an incubator The following are desirable attributes of an incubator, unrelated to specific requirements, such as low temperature control or hypoxia. Many of these relate to cleaning the incubator and it must be emphasized that this is done regularly.

Interior corrosion-resistant, for example, stainless steel. Copper will give better control of fungal contamination but does tend to corrode. Copper alloy (antimicrobial copper) may be a suitable alternative but is expensive.

Smooth interior with no crevices; easy to access and clean.

Shelving/racking that can be removed completely for cleaning and sterilizing if necessary.

Dual temperature control with safety override.

Although some suppliers provide HEPA-filtered air circulation, non-fan-assisted incubators will reduce the airborne spread of contamination within the incubator and are generally preferable.

With modern design and insulation a water jacket is not required and makes the incubator difficult to move. It does, however, hold its temperature for a longer period if the power fails.

Large incubators should be avoided; it is better to have two or more smaller incubators. It makes cleaning easier and gives better back-up in the event of failure or contamination.

Vibration free, as vibration can cause non-random distribution of cells.

Humid atmosphere with CO 2 controlled by non-corroding sensor, for example, gold wire or infra-red.

Data output ports to record temperature and CO 2 fluctuations.

A cooled incubator should be used if it is to be located in a warm laboratory.

Controlling contamination Microbial contamination in cell culture humidified CO 2 incubators can be minimised by following a number of simple preventative steps including: (1) good aseptic technique; (2) using incubators with design features to reduce contamination (Section 3.4.2) having a regular cleaning strategy together with a periodic decontamination method.

Aseptic technique

Cell culture vessels are a major source of contamination and should be wiped with 70% alcohol before being placed in the incubator.

Where possible cell culture flasks with secure 0.22- μ m filter caps should be used in preference to loose-lidded dishes and plates, to prevent spills and aerosols of cell culture media that will allow microorganisms to grow.

Regular cleaning and maintenance

The shelves, inner and outer doors and door seals should be wiped down in situ with a suitable disinfectant cleaner followed by 70% alcohol, to remove any residue, once a week. Any visible spillages of medium should always be immediately removed.

Water in the humidity tray should be replaced weekly and the tray cleaned as above. Adding disinfectants or chemicals to the water in the incubator humidity tray is not recommended as many of these will produce volatile compounds, which may be cytotoxic to the cultured cells. Instead sterile distilled water should be used and replaced weekly. However, many laboratories do successfully use a number of chemical additives in their incubator humidity trays including copper sulphate (1.0 g per litre) and a number of commercially available products containing mainly quaternary ammonium compounds such as Aqua Clean ( www.cleantabs.co.uk ), Clear Bath ( www.spectrumlabs.com ), SigmaClean ( www.sigmaaldrich.com ) and Aquaguard ( www.promokine.info/products ).

All air, CO 2 and HEPA filters should be changed at the appropriate service intervals, and door seals and gaskets replaced if showing signs of wear.

Suggested decontamination procedure

Should be performed immediately if cell cultures are found to be contaminated and at regular weekly, monthly or quarterly intervals depending on the type of work being conducted.

Switch the incubator off.

Remove all metal shelving, brackets and supports from the incubator. Wash with a suitable detergent, allow to dry and then autoclave. Copper shelving is quite soft and may warp in the autoclave but can be wiped down with a suitable disinfectant followed by 70% alcohol rather than autoclaving.

Remove the humidity pan, if separate, discard the water and wash and disinfect the pan as above.

If the humidifying water sits in the bottom of the incubator, drain and then wipe down all internal surfaces with a suitable detergent. Allow to dry and then wipe down with a suitable disinfectant followed by 70% alcohol, making sure not to damage internal sensors or fans.

Remove the door seal, if possible, then clean and disinfect it as above.

Reassemble the incubator, switch on and allow it to reach working temperature and CO 2 concentration before use.

Many of the CO 2 incubators currently available have built-in automatic high temperature decontamination cycles, which will usually either be moist heat at 90 °C or dry heat at 180 °C. These processes run overnight and aim to eliminate the need to remove and manually disinfect the incubator components. If available the decontamination cycle should be run immediately if any cell cultures are found to be contaminated. Be aware that if a fungal contaminant is present you will need to run two decontamination cycles with a 12-h gap. The first will kill any vegetative organisms and any resistant spores will then germinate and be killed in the second cycle. Automatic decontamination cycles can also be run as a preventative measure at weekly, monthly or quarterly intervals depending on the type of work being conducted.

Abbreviations

Advisory Committee on Dangerous Pathogens

Advanced Therapy Medicinal Product

American National Standards Institute

American Type Culture Collection

Biological Safety Committee

bovine pituitary extract

bovine spongiform encephalopathy

British Standards Institute

Convention on International Trade in Endangered Species

Committee for Advanced Therapies

central nervous system

copy number variation

Control of Substances Hazardous to Health

Department of Agriculture, Fisheries and Forestry

double distilled water or distilled deionized water

Department for Environment, Food and Rural Affairs

Dulbecco’s modification of Eagle’s medium

dimethyl sulphoxide

Deutsche Sammlung von Mikroorganismen und Zellkulturen

Epstein Barr Virus

European Collection of Cell Cultures

European Medicines Agency

European Union

Food and Drug Administration (USA)

Good Cell Culture Practice

Good Clinical Practice (Laboratory)

Good Laboratory Practice

Good Manufacturing Practice

human embryonic stem cell

Human Fertilisation and Embryology Authority

human immunodeficiency virus

Health and Safety Executive

Human Tissue Authority
Human Tissue Act

human T-cell leukaemia virus

International Air Transport Association

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use

Investigational Medicinal Product

induced pluripotent stem cell

Integrated Research Application System

International Union for Conservation of Nature

in vitro fertilisation

Japanese Collection of Research Bioresources

leukemia inhibitory factor

liquid nitrogen

mouse embryo fibroblast

Medicines and Healthcare Products Regulatory Agency

Medical Research Council

microbiological safety cabinet

material transfer agreement

National Health Service

National Research Ethics Service

polyvinylidene fluoride

quality control

research ethics committee

selenium, insulin and transferrin

standard operating procedure

specific pathogen free

short tandem repeat

tetradecanoylphorbol acetate

total organic carbon

transmissible spongiform encephalopathy

UK Stem Cell Bank

ultrapure water

World Health Organization

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Role and relevance of fish cell lines in advanced in vitro research

Published: 11 January 2022
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M. Goswami ORCID: orcid.org/0000-0001-6863-7647 1 ,
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Vance Trudeau 2 &
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Introduction

Cell line derived from fish has been established as a promising tool for studying many key issues of aquaculture covering fish growth, disease, reproduction, genetics, and biotechnology. In addition, fish cell lines are very useful in vitro models for toxicological, pathological, and immunological studies. The easier maintenance of fish cell lines in flexible temperature regimes and hypoxic conditions make them preferable in vitro tools over mammalian cell lines. Great excitement has been observed in establishing and characterizing new fish cell lines representing diverse fish species and tissue types. The well-characterized and authenticated cell lines are of utmost essential as these represent cellular functions very similar to in vivo state of an organism otherwise it would affect the reproducibility of scientific research.

The fish cell lines have exhibited encouraging results in several key aspects of in vitro research in aquaculture including virology, nutrition and metabolism, production of vaccines, and transgenic fish production. The review paper reports the cell lines developed from fish, their characterization, and biobanking along with their potential applications and challenges in in vitro research.

Fish cell line: depositories, web resources and future applications

Applications of Fish Cell Cultures

ABCD of Zebrafish Culture

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The Development of chemically defined cell culture medium like Leibovitz -15 (L-15) and the development of antibiotics with gradual improvisation of cell culture techniques eventually made the generation of cultured cells for deriving continuous cell lines. In addition to being an important biomedical tool like any other cell line, cell cultures prepared from fish, shellfish and seaweeds can provide a significant contribution to the growth of aquaculture. The scientific knowledge gained through the cell culture system can be utilized for manipulating the whole organism to enhance its usefulness for aquaculture. Their cell line could be useful for providing basic insights into growth, reproduction, and health, creating opportunities for manipulation and thus the cell lines could be used as sources of biochemical products in place of the whole organism [ 1 ]. Cell-based aquaculture systems using cell cultures could be a game-changing practice to produce seafood and other aqua food across multiple species for meeting the demand of the burgeoning world population [ 2 ]. A cell-based aqua food production system utilizing cells in place of whole fish could also lead to greater preservation of the aquatic environments. This practice has to meet the regulatory framework/guidelines developed by the FDA [ 3 ] for the safety of food produced using such animal cell culture technology.

Fish cell culture offers several advantages over mammalian cell culture in terms of adaptation to a broad range of temperature, higher tolerance to hypoxia, easier maintenance of cell culture for longer periods. Cell lines from fish have been increasingly established from different aquaculture species and they are being used in in vitro research related to aquaculture and other interdisciplinary areas. However, there are emerging issues regarding standardization of cell line nomenclature, characterization of cell lines following SOP/recommended guidelines, and conservation of cell lines in separate biobanks across the world-which we review below is of utmost essential to maintain scientific reproducibility in cell-based biological research using fish cell lines. The key areas of aquaculture like fish health, disease diagnosis, safety, and nutritional aspects challenging aquaculture production can be studied using fish cell lines without scarifying whole live fish (Fig. 1 ). The scientific knowledge generated using fish cell lines would be immensely useful for quality fish production in a sustainable manner. Cell lines would facilitate in vitro research for developing climate-resilient and sustainable aquaculture systems to minimize the key challenges and provide nutritional security to the burgeoning world population.

Implications of fish cell line in aquaculture

Global status of fish cell lines

An increasing trend has been observed for the development of fish cell lines from a wider range of tissues covering both tropical and temperate water since the first establishment of the RTG-2 cell line in 1962 [ 4 ]. Bairoch enlisted 883 fish cell lines out of 104,421 cell lines from > 590 species in Cellulosaurus; a knowledge resource on cell lines [ 5 ]. In general, cell lines have been developed globally using different types of fish tissue samples including gill, caudal fin, eye, liver, and kidney. Fish cell lines have also been established using tissue samples like intestine [ 66 ], brain [ 95 ], vertebra [ 105 ], and snout [ 121 ]. Spontaneous differentiation is one of the most challenging for the development of embryonic stem cell culture from fish and this is the main cause behind a very limited number of stem cell lines. Few embryonic fish stem cell lines were developed from sea bream Sparus aurata [ 6 ], sea perch Lateolabrax [ 7 ], sea bass; Lates calcarifer [ 8 ], Catla catla [ 9 ], Labeo rohita [ 10 ]. A feeder-free cell culture system used for the development of Embryonic Stem (ES) cell lines from medaka and zebrafish has boosted fish stem cell research by replacing the use of feeder layers to inhibit spontaneous differentiation in fish stem cell culture [ 11 ].

Cell line characterization

The numbers of cell lines developed from fish have been increasing rapidly which raises the concern for accurate authentication and characterization of fish cell lines to provide reproducible scientific data. The comprehensive guidelines for using cell lines highlight various aspects of cell culture, issues of misidentification, contamination with microbes along with recommendations to overcome these problems [ 12 ]. Although these guidelines are meant for scientists in the UK, the basic principles remain the same for international implications. Research and development using cell lines need detailed knowledge on the purity and originality of the cell line [ 13 ]. The characterized cell lines are indispensable as they facilitate the researchers to perform in vitro research and standard guidelines are available for their characterization. However, many fish cell lines don’t meet uniform international standards. The Food and Drug Administration has described the steps to be considered while characterizing a cell line used to produce biological products [ 14 ]. Such standard protocol for the characterization and authentication of fish cell lines should be practiced throughout the world. Standard protocols for authentication of cell line have been reported [ 15 ] wherein standard methods like cytochrome c oxidase subunit1 (CO1) barcode, Short Tandem Repeat (STR) profiling, karyotyping, Single Nucleotide Polymorphisms (SNP) profiling, use of species-specific primers, whole-genome sequencing (WGS), etc. are described as ideal approaches for authentication and maintenance of quality cell lines. Several STR databases of cell lines are maintained by ATCC, DSMZ across the world. CLASTR: The Cellosaurus STR similarity search tool is now in the public domain for comparing STR profiles of the cell lines [ 16 ]. Cross-contamination also causes a disastrous feature of the cell line as the cell line losses its originality and hence cross-contamination needs to be avoided by following standard operating procedure (SOP). Development of a framework for cell line annotation linked to STR and SNP profiles in the form of a catalog of synonymous cell lines to avoid or detect cross-contamination [ 17 ].

Misidentification of cell lines leads to irreproducible data and hence proper authentication of cell lines using molecular markers is essential. It was obligatory to provide DNA-based certification of the cell line developed [ 18 ]. Mitochondrial DNA genes like 16S rRNA and CO1 are used for the authentication of cell lines. Cox I gene has been used as a molecular identification system for animal species which is popularly referred to as ‘‘DNA barcoding’’ [ 19 ]. The cox I gene was used as DNA barcodes for the authentication of 67 cell lines [ 20 ]. Similarly, many fish cell lines have been DNA barcoded using cox I [ 21 , 22 , 23 ]. Cell line repositories like DSMZ, ATCC use DNA barcoding as a standard method for cell line identification. Protein expression signature has also been used for the identification of cell lines derived from fish [ 24 , 25 ].

Cell lines developed from fish are mostly applied in basic, biomedical and toxicological research in addition to their potential applications in aquaculture. Several key issues in aquaculture can be addressed by cell culture technology and they are reviewed below.

Fish health management

The fish disease has been considered as one of the most critical challenges for sustainable aquaculture production due to the economic loss and widespread use of antibiotics and other compounds causing great risk to the aquatic environment. Fish cell culture has great potential to provide tools and strategies for disease control in aquaculture. In vitro models that use cell culture methods and experimental systems facilitate a deeper understanding of the complex interactions underlying disease outbreaks and its advancement in which the interactions between aggressors and the host can be dissected [ 26 ]. Fish cell lines have potential applications in understanding disease mechanisms, developing assays for disease diagnosis, developing drugs and vaccines for the control of the fish disease. The export trade of seafood depends upon the quality and health status of the seafood. The fish cell line model has been considered useful for detecting viral pathogens and strategies need to be implemented accordingly for the health protection of major aquaculture species. Zoonotic disease associated with fish is another concern where consumption of unhealthy fish might be a risk to a human being. Associated in vitro assays would be useful in detecting such harmful pathogens and allergens so that the quality of seafood can be augmented. In vitro methods using cell cultures for addressing health issues in molluscs and crustaceans are equally important. Department of Biotechnology, Govt. of India has funded a national programme on the isolation and characterization of finfish and shellfish viruses using cell lines in India. In vitro approach using permanent cell lines needs to be validated for fish and shellfish disease surveillance and health certification. Transboundary movements of live aquatic animals have greatly increased concern for spreading disease in the aquaculture system.

The viral disease used to cause devastating loss to the aquaculture industry. The entire world has witnessed the deadliest effect of the spread of the virus Covid-19. The isolation of the novel Covid-19 using the animal model has begun and the successful isolation would be useful in understanding the biology and evolution of the Covid-19 in developing drugs, vaccines, and rapid diagnosis kits. Isolation of viruses using fish cell lines is one of the most sensitive techniques for the discernment of the important pathogens causing viral disease in many fish and other species. Hence, the development of control measures to halt the spread of the viral disease depends on the unitisation of fish cell lines for such purposes. A comprehensive list of fish cell lines used in virus susceptibility studies is given in Table 1 . Research on the avoidance of infectious fish disease in aquaculture necessitates a cell culture- based approach for understanding the underlying disease mechanism. Fish cell culture-based isolation and propagation of virus has provided momentum to virological studies and facilitated research on viral diseases in important aquaculture species. Propagation of viruses in a cell culture system is one of the bases of a virus surveillance system using cell culture. Ariel et al. developed standard methods to reduce false negatives in cell culture-based surveillance systems in testing fish cell line susceptibility for the viruses [ 27 ].

Highly specific cell lines are used for investigating unique virus which otherwise doesn’t propagate in any normal cell line. The susceptibility of the fish cell lines to the virus varies with species as well as tissue from where the cell line is developed . This raises the importance of the development of species-specific and tissue-specific cell lines from various important aquaculture species. Some fish cell lines like bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprinid (EPC), fathead minnow (FHM), rainbow trout gonad (RTG-2), and SAF-1 have shown susceptibility to some of the most commonly available viruses like Infectious pancreatic necrosis (IPN), VHSV, IHNV, IPNV, SVC, koi herpesvirus (KHV) and Channel catfish virus (CCV) that have severely affected several aquaculture species [ 28 , 29 ]. MEF-8C1 cloned cells obtained from the MEF cell line from mandarin fish suitably propagated megalocytiviruses that cause major problems in finfish aquaculture in China [ 30 ]. A transgenic fish cell line RTG-P1 was applied to estimate viremia of Salmonid alphavirus (SAV) which causes a serious viral disease i.e. Salmon Pancreas Disease (SPD) in Atlantic salmon farming [ 31 ]. SISK and SISS cell lines developed from the kidney and spleen of Lates calcarifer respectively and SIGE cell line developed from the eye of Epinephelus coioides showed their ability to propagate a nodavirus strain [ 32 ]. Yashwanth et al. reported the susceptibility of the OCF cell line to NNV [ 23 ]. SSN-1 cell line supported the replication of snakehead fish vesiculovirus (SHVV) which causes great economic loss in fish culture in East Asian countries [ 33 ]. Understanding the transmission of viral infection between the two most important aquaculture species mandarin fish and snakehead fish, it would be useful to develop control measures to prevent the spreading of such viral diseases. Fish cell cultures or cell lines could be used for investigating viral pathogenesis and host–pathogen interactions.

In the past, such in vitro methods were used for some bacterial pathogens like mycobacterial host–pathogen interactions using the carp monocytic cell line CLC (carp leukocyte culture) [ 34 ]. Recently, Cardiac Primary Cultures (SCPCs) from Atlantic salmon pre-hatch embryos were used to investigate viral host–pathogen interactions and pathogenesis [ 35 ]. A blend of cell culture and molecular biology methods will provide deeper insights into host–pathogen interactions. Although advanced antibody-based techniques are being developed in disease control in aquaculture, fish cell culture continues to be an indispensable technique for isolation and characterization of the pathogenic virus and intracellular bacteria and studying their pathogenicity [ 26 ]. These fish cell lines are going to play a crucial role in virus isolation and understanding viral pathogenicity and thereby controlling these viral diseases to enhance sustainable aquaculture production.

Pathological & immunological studies

Several fish health-related issues can be studied in vitro using fish cell lines. The most prominent is the application of fish cell lines in disease diagnostics and immunological studies. Some intracellular bacterial fish pathogens like Rickettsiae spp and Renibacterium salmoninarum have been detected in fish using cell cultures [ 36 , 37 ]. The in vitro investigation using fish cell line (CHSE-214) has improved the knowledge of the infection process by Yersinia ruckeri in salmonid fish as well as the interaction between the pathogen and host cells [ 38 ].

Cell cultures are promising in vitro tools in studying the host defense mechanism and thereby help in exploiting the immunological information for the health protection of fish and shellfish used in aquaculture. Fish leukocyte cell lines and macrophages developed from many aquaculture species like carp, catfish have been used for immunological studies. Several monocyte-like cell lines have also been developed using peripheral blood leukocytes of channel catfish [ 39 ]. Cell lines developed from gut, skin, and gill are promising in vitro tools for studying the defense mechanism in fish. The immunological potentials of DNA vaccines, synthetic peptides and immunostimulants, and other products can be tested using these fish cell lines. A continuous blood cell line developed from peripheral blood mononuclear cells of Cyprinus carpio was useful in understanding the fundamental aspects of fish immunology [ 40 ]. Fish macrophage cell lines are found to be very useful in numerous research applications including immunological studies. Two macrophage cell lines i.e., CTM and CCM [ 41 ] developed from Catla catla could be useful in investigating the importance of these cell lines in the differentiation and maturation of thymocytes and other fish immunological studies. SHK-1 macrophage-like cell line developed from Atlantic salmon showed the reaction to monoclonal antibodies against Atlantic salmon peripheral blood leukocytes and the cell line was able to phagocytose bacteria [ 42 ]. Macrophage-like cell line RTS11 developed from rainbow trout was used as a promising tool for investigating immune cell-specific responses in vitro [ 43 , 44 ].

Saprolegniales are considered the most important fungi causing disease in freshwater fish. The cytological response of their piscine hosts is not precisely understood. RTS11 cell line developed from rainbow trout was used to check the response of macrophage to water molds Achlya and Saprolegni [ 45 ]. Fish cell lines are a very useful aid in understanding pathogenicity arising due to nutritional issues. Such studies were carried out using fish cell lines to investigate the proinflammatory mechanism underlying the relationship between dietary PUFA and cardiac lesions using a cell line developed from chum salmon [ 46 ].

gene-editing and genetic engineering

Genetically edited fish cell lines have enormous biotechnological and clinical applications. CRISPR (Clustered Regularly Interspaced palindromic repeats-Cas9 (CRISPR associated) has revolutionized gene editing. Generation of improved fish cell lines using CRISPR-Cas9 technology would facilitate aquaculture biotechnological research including fish disease studies. Genetically edited cell lines using genome editing technology would be useful to enhance the transfection efficiency of fish cell lines and utilize those cell lines for the efficient production of viruses for vaccine development. This technique has been mostly used for gene editing in mammalian cell lines whereas the use of gene editing for fish cell lines is in the infancy stage. The use of CRISPR-Cas9 based gene editing method has been reported earlier in fish but an efficient method for gene editing was developed in a fish cell line CHSE developed from Chinook salmon Oncorhynchus tshawytscha for the first time [ 47 ]. The cell line was genetically engineered to overexpress different forms of CHSE cell line. Although various attempts have been made, a convincing fish knock-out in vitro model has not yet been developed.

A stable trout head kidney cell line was transfected with a variety of plasmids expressing cytokines Interleukin-6 macrophage colony-stimulating factor (MSCF). Rainbow trout head kidney cell line and RTG-2 stable cell lines were engineered in developed conditioned media to express interleukin (IL-2), IL-6, and macrophage colony-stimulating factor (MCSF) [ 48 ]. Greasy grouper Epinephelus tauvina liver cell line GL-av was genetically modified to assess the effectiveness of the anti-apoptotic protein Bcl-xL [ 49 ]. Fish cell lines have also important applications in in vitro ploidy manipulation. Polyploidization was successfully obtained in a crucian carp induced by a chemical compound and developed an autotetraploid cell line [ 50 ].

Genetically engineered fish cell lines have enormous potentials to be used in fish health, genetics, and biotechnological research. The establishment of a stable cell line is the need of the hour for functional genomics studies for fish genetics and health. With the progress of gene delivery methods, the number of stable genetically modified fish cell lines has increased. Not much effort has been made for the functional characterization of immortal fish cell lines towards developing genetically engineered methodologies [ 51 ]. Genetical modification of goldfish cell line, Chinook salmon Oncorhynchus tshawytscha embryo CHSE cell line, rainbow trout Oncorhynchus mykiss hepatoma RTH cell line have provided interesting information for fish disease and immunological research [ 52 , 53 ]. A transformed EPC (Epithelioma Papulosum Cyprini) cell line under the control of the tilapia HSP70 promoter expressed a green fluorescent protein (GFP)-luciferase fusion gene in response to cellular stress [ 54 ]. The fish cell lines can be used for studying stressors concerning infectious fish disease in addition to their usage in investigating environmental stressors concerning climate change. A novel in vitro system was developed using genetically modified Chinook salmon embryonic (CHSE)-TOF cell line to measure the sensitivity of some important virus-like Infectious Haematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV), Salmon Alphavirus (SAV), and Epizootic Haematopoietic Necrosis Virus (EHNV) [ 55 ].

Transgenic studies and reproductive biotechnology

Gene targeting and transfer of the genes for transgenic fish production become easier with the advancement of cell culture techniques. Transgenic zebrafish produced applying primary cultures of genetically modified zebrafish male germ cells has paved the way for the development of transgenic lines in model organisms or other animal systems [ 56 ]. Genetically modified myogenic cell culture was developed from a transgenic trout ( Oncorhynchus mykiss ) having a construct containing the GFP reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter [ 57 ]. The transgenic line can be produced by utilizing the primordial germ cell (PGC) cultures. Vasa marker facilitates isolation and characterization of targeted PGCs for germline-specific expression in fish. Tanaka et al. developed a transgenic line of medaka using GFP expressed germ cells [ 58 ]. Successful transplantation of germ cells in fish demonstrated the possibility of surrogate broodstock production in the aquaculture system. Intraperitoneal transplantation of PGCs was used to produce seedlings in rainbow trout for the first time [ 59 ]. The progress in stem cell culture and their subsequent applications in vitro basic research, as well as aquaculture biotechnology, will transform the fisheries sector for achieving the blue revolution. Spermatogonial stem cells transplantation offers many scopes for a successful captive breeding programme for aquaculture species. A spermatogonial cell line (SG3) developed from the mature testis of medaka was capable of producing sperm [ 60 ]. The production of fertile medaka fish using ES cells proved the possibility of generating nuclear transplants using fish embryonic cells [ 61 ]. More research needs to be carried out in aquaculture species utilising these ES cells. Gene transfer through embryonic stem (ES) cell is a promising tool for the production of transgenic animals [ 62 ]. Yoshizaki et al. successfully developed a stem cell-mediated gene transfer method to produce transgenic rainbow trout [ 63 ]. ES cells along with PGCs and nuclear transfer strategy make the transformation efficient for transgenic fish production.

Fish cell lines as in vitro models

Fish cell lines have enormous potentials to be used model systems for studying fish disease, immunology, biotechnology, nutrition, and toxicity testing of chemicals and therapeutic agents used in aquaculture as they are ideal substitutes for the whole organism which involves increasing questionable ethical issues. In vitro model has been used for investigating the viral replication and genetics and the production of experimental vaccines to be used in aquaculture. Organ culture developed from tilapia, eel, and trout pituitary glands was used as in vitro model for the production of the growth hormone prolactin [ 64 ]. Fish cell lines were found to complement in vivo development studies and recognize the involvement of signaling pathways in the developmental processes [ 65 ].

Cell cultures developed from fish can be effectively used as model systems to investigate nutrient assimilation and metabolism in fish but rarely such a culture system has been used to study that aspect of fish nutrition. This also raises the need for the development and characterization of the intestinal cell culture systems to support such studies. Cell line developed from the fish intestine is useful in understanding the effect of functional feed ingredients like probiotics and dietary exposure to chemicals in the aquatic system. Kawano et al. reported the use of the intestinal rainbow trout epithelial cell line (RTgutGC) to elucidate the metabolism of environmentally relevant contaminants in the intestinal tract of fish [ 66 ]. Recently, RTgutGC was used as an in vitro model for understanding the functional immunity system of the fish gut as well as the effects of functional feed ingredients in the gut cells [ 67 ].

Langan et al. investigated the function of spheroid size in the metabolism of propranolol using an RTgutGC cell line as a 3D fish intestinal model [ 68 ]. The cell line of the intestinal epithelial region rainbow trout acts as a barrier to study cellular mechanism of immune function, physiological and pathological response, nutrient uptake, and toxicants [ 118 ]. The above RTgutGC cells were compared with new cell lines from the proximal and distal intestine of rainbow trout such as RTpi-MI & RTdi-MI and these cells formed a polarized barrier, which was not permeable to larger molecules and absorbed glucose and proline [ 119 ]. The RTgill-W1 cells were used as in vitro model for accessing acute toxicity of select chemicals associated with Whole Effluent Toxicity (WET) testing in both marine and freshwater conditions [ 120 ]. A physiologically realistic model system- fish-gut-on-chip was developed by combining intestinal cell culture from rainbow trout ( Oncorhynchus mykiss ) with microchip technology and microfluidic engineering to study its barrier function towards the environment i.e. food & water and to monitor the function in real-time [ 69 ]. In vitro models are extremely important to study collagen synthesis and secretion in humans and other higher vertebrates. Very few models have been established to investigate collagen synthesis and secretion in fish. Lee and Bols reviewed the potential applications of fish cell lines to study collagen as in vitro model for evaluation of physic-chemical factors controlling synthesis, secretion, and deposition of collagen [ 70 ].

Cell-based aquaculture

Aquaculture has been growing very fast and facing several challenges to meet the rising demand ensuring the safety and quality of fishery products. The concept of producing cell-based seafood has been emerging as a new approach to producing alternate animal protein. This alternative approach of animal protein production from fish would address several key challenges faced by the conventional aquaculture systems and declining marine capture fisheries. This alternative fish production system will reduce pressure on natural resources and the environment. Accordingly, the entire world is moving towards climate-resilient production systems and in vitro meat production has emerged as an area of cutting edge and priority research. The successful launch of the in vitro hamburger in 2013 has accelerated the research focus on cell-based meats [ 71 ]. The ease of growing fish cells at a lower temperature compared to mammalian cells may give cost benefits to the production of cellular fish meat as compared to cellular animal meat. Tissue engineering blend with modern aquaculture techniques can be explored to utilize marine cell culture as an attractive opportunity for the production of in vitro fish meat. Fish muscle cell culture can be used for in vitro fish meat production by exploiting their salient physiological properties like tolerance to a hypoxic-conditions, high buffering capacity, and lower temperature [ 2 ]. Fish muscle cell cultures are more adaptable to in vitro conditions than mammalian ones and hence in vitro meat production will be more feasible with fish muscle cell cultures. More concerted efforts and investigations are required to generate information on fish and shellfish muscle cell culture systems to suit in vitro fish meat production systems. The fastest possible path to produce cellular fish meat should start with zebrafish for research and development purposes [ 72 ].

The importance of genetic modification and closed aquaculture system paves the way for the innovative concept of cell-based fish production i.e. cellular aquaculture [ 73 ]. American space organization NASA had supported the first research program on in vitro edible muscle protein production from goldfish for space travelers during long-term manned space exploration [ 74 ]. A better understanding of the myogenesis involved in the muscle cell and tissue culture would be essential to trap the benefits of muscle cell culture in promoting cellular aquaculture. In vitro models like C2C12 cell lines have been utilized in understanding molecular mechanisms underlying muscle growth and differentiation in mammals [ 2 ]. Such studies are in the infancy stage in teleost due to the unavailability of equivalent permanent muscle cell lines except for a few fish muscle cell lines [ 75 , 76 , 77 , 77 ]. Most of them are not from aquaculture species except muscle cell lines developed from Wallago attu [ 21 ], olive flounder Paralichthys olivaceus [ 78 ] and some myosatellite cells developed from the primary culture of muscle-derived from carp [ 79 ] and rainbow trout [ 80 ]. Prospects of cell-based aquacultures will rely on the development of appropriate cell lines, optimization of growth media, and other factors, mass production of cells. Some institutes like Good Food Institute; New York, USA have taken initiatives to develop cell-based seafood.

The cell-based molecular mechanism studies will provide basic research data for cell-based fish production. Some investigations on harvested native muscle tissues from fresh water and marine fish provide interesting insights into the potentials of developing a muscle cell culture system [ 81 , 82 ]. The genome editing by CRISPR/Cas9 system is a promising tool that attains targeted gene editing with high efficiency, without the requirement of integrating an exogenous gene. Its potential is yet to be exploited much in aquaculture using fish cell lines. CRISPR/Cas9 system has been used to get higher skeletal muscle/ muscular growth in aquaculture species like red sea bream; Pagrus major [ 83 ] and channel catfish; Ictalurus punctatus [ 84 ]. Clean meat farm is a million-dollar industry but academic research lags to propagate clean meat production [ 85 ]. Academic research focusing on the development of muscle cell culture systems, standardization media, and bioreactor facilities for large-scale cell production would be required to accelerate in vitro fish meat production and bring it to market.

Vaccine and other products developed from fish cell culture

Global aquaculture particularly shrimp farming used to suffer a major economic loss every year due to the occurrence of viral diseases. The development of vaccines has great relevance to the aquaculture industry to mitigate viral diseases. Purified viruses are likely to be the first health product for use as vaccines obtained from piscine cell cultures [ 1 ]. Several viral vaccines have been produced with improved techniques for their delivery at affordable prices [ 86 ]. Several fish cell lines have been tested for virus replication towards vaccine development. There is a need for scaling up the efforts towards the development of effective vaccines.

Cell-culture-based technology can be used as a robust and reliable alternative for the production of vaccines. The development of the vaccine and its potency testing requires a large number of live fish. Fish cell culture can be used as an alternative to whole live fish for the production and testing efficacy of fish vaccines. Cell lines like Vero, Madin Darby canine kidney (MDCK), chicken embryo fibroblasts (CEFs) have been mainly used for viral vaccine production [ 87 ]. Cell lines developed from humans, monkeys, hamsters, dogs, and chickens have so far been used for the development of vaccines. The studies for the development of viral vaccines using fish cell lines are very much limited. Oh et al. reported that the formalin-inactivated RSIV vaccine was developed from the viruses propagated in Grunt Fin (GF) cells [ 88 ]. Several inactivated or attenuated fish viral vaccines have been developed for iridovirus and NNV protection [ 89 , 90 ], and some of them have been commercialized [ 91 ]. However, few cell lines are available to replicate megalocytivirus, betanodavirus, herpesvirus, and aquareovirus for vaccine production, and hence more efforts are warranted to develop specific cell lines for the proliferation of these viruses. Fish cell cultures have great applications in modern vaccine technology including recombinant, DNA/RNA particle vaccines. Only a few fish cell lines have been used in viral propagation leading to vaccine development and diagnostics, and many are under trial for vaccine production. Rainbow trout pronephros cells as in vitro model could be used to screen fish DNA vaccine [ 92 ]. The anti-VP5 polyclonal antibody was able to neutralize Grass carp reovirus (GCRV) through in vitro micro neutralizing assay in a grass carp cell line CIK [ 93 ]. This would be important towards the development of a vaccine to prevent the infection of GCRV in grass carp which causes great damage to grass carp production in China.

The deficiency of treatment options and limited availability of vaccine poses a challenge for control of viral disease control in aquaculture. In this regard, JL122, a broad-spectrum antiviral agent oxazolidine compounds, was proven to inhibit transmission of IHNV, VHSV, and SVCV in the EPC cell line [ 94 ]. Another small molecule LJ001, lipophilic thiazolidine derivative also showed broad-spectrum antiviral properties for inhibition of IHNV infection in the EPC cell line. These hold promise as an immersion treatment option for the outbreak of aquatic rhabdoviral infection. The complex interaction between Infectious kidney and spleen necrosis (ISKNV) and its host Chinese Perch Brain (CPB) cells generated new information for understanding viral pathogenesis and developing antiviral treatment strategies [ 95 ].

Fish cell lines exhibit less transfection efficiency, unlike mammalian cell lines. Low transfected cell lines are not useful for the production of recombinant protein and other products. In the case of mammalian cell lines, a higher range of transfection efficiency of mammalian cell lines with the aid of the right combination of cell type and method was achieved up to 100% [ 96 ]. However, the same methods applied to fish cell-cultured at lower temperatures (5–15 °C) provided the low transfection efficiency which is often below 10% [ 97 ] whereas the transfection efficiency of the head kidney cell line was improved from 11.6% to 90.8% using Amaxa's cell line nucleofector solution T and program T-20 [ 98 ]. Hence, alternative reagents or methods should be explored to enhance transfection efficiency in fish cell lines. In addition to vaccine production, fish cell lines should also be explored for the production of human pharmaceutical proteins. The ability of the fish cells to grow at as lows as 4 0 C could be exploited in this regard. Transformed fish cell line Epithelioma papulosum cyrpini cells (EPC) were used to stably express and secret recombinant pleurocidin (Ple), a linear cationic peptide of 25 amino acids uninterruptedly for more than 2 years [ 99 ]. Fibroblast cell plays an important role in increasing collagen synthesis, collagen secretion under the stimulatory influence of ascorbic acid. Some cell lines developed from fish have been reported to be an ideal in vitro source for the synthesis of collagen [ 100 ]. Cytokines such as interferon could be considered for their potential therapeutic potential to fill the gap of shortage of fish therapeutics [ 1 ]. Fish interferon was partially purified in small quantity from the rainbow trout gonadal cell line, RTG-2. Transfected RTG-2 cell line expressed Interleukin Cytokines (Interleukin (IL)-2, IL-6, and Macrophage Colony Stimulating Factor (MCSF) and the transfected cell line was used to produce conditioned media-rich in these cytokines [ 48 ].

Toxicological and environmental monitoring studies

Different inorganic and organic aquatic pollutants influence the quality and health status of farmed fish and shellfish. Proper investigation to know the ill effects of the aquatic pollutants on farmed fish and shellfish is the need of the hour to improve the marketing of quality seafood. The cell lines have been used as alternative tools to replace the use of whole live fish due to a significant correlations observed between in vitro and in vivo data. Cell lines have been applied as a rapid and economic in vitro tool for screening toxicity of chemicals and environmental samples [ 1 , 93 ]. Fish cell lines have important applications in studying the effects of different aquatic pollutants on the metabolism of aquatic biological systems and hence there is a potential application of fish cell lines in environmental monitoring. Fish cell lines have been adopted as an in vitro tool for ecotoxicological evaluation of chemicals by many international regulatory bodies like Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) in Europe, Food, and Drug Administration (FDA) in the USA. Fish cell cultures facilitated in vitro investigation to find toxic effects of polycyclic aromatic hydrocarbons and aflatoxins in farmed fish [ 101 ]. Primary cultures have been used in the case of toxicological investigation in invertebrates as permanent invertebrate cell lines are not available. Considerable progress has to be made for the development of invertebrate cell lines to facilitate in vitro investigations in farmed shelf fish and mollusc. In addition to the aquatic pollutants, toxic and residual effects of antimicrobial drugs used in aquaculture need to be investigated where fish cell culture can be utilized to replace the whole live fish model. In vitro studies established a correlation between in vitro immunosuppression and the interference of various antimicrobial drugs [ 102 ]. In this regard, in vitro investigation will provide more insights to increase the awareness of global antimicrobial resistance (AMR) initiated World Health Organization.

Fish nutrition and metabolism

Fish cell lines have the potentials to be used in fish feed formation using alternative ingredients as they provide an excellent in vitro model to study nutrient absorption and assimilation. To facilitate such studies, more intestinal fish culture systems need to be developed. Due to the lack of targeted research tools, the current understanding of the underlying effects of feed ingredients on fish nutrition is limited. The application of appropriate fish cell lines would facilitate further research on the basic functions of the digestive tract and the effects of functional feed ingredients on various aspects of fish nutrition [ 67 ]. The vital role of cell lines in biological experimentations is to reduce animals, with major three R rules such as reduction, replacement, and refinement [ 103 ]. That enhances the interest of researchers to utilize the in vitro model to study cellular environmental conditions of living biological components. The primary cultures of adipocytes or hepatocytes and myoblasts were significantly used to study molecular mechanisms related to fish nutrition [ 104 , 105 , 106 , 107 , 108 ]. This approach provides significant progress to a limited extent because the primary cultures failed to allow the functional genomic analysis to study the specific gene functions.

Morin et al., 2020 studied the role of RTH-149, RT hepatoma-derived cell line to address nutrition-related queries based on major pathways such as macroautophagy (autophagy), general control nonderepressible 2 (GCN2), and mechanistic Target of Rapamycin (mTOR) pathway that regulate cell homeostasis through amino acids to study the nutrient-sensing signalling. These pathways had attention concerning rainbow trout nutrition, which strongly relies on the supply of amino acid and assessing (1) their capacity to be repressed or induced by starvation, (2) their specific regulation by amino acid availabilities, and (3) their related kinetics. They demonstrate that the starvation can be sensed by RTH-149 cells, which then induce the activation of GCN2 and drive the expression of ISR-related genes in an amino acid-dependent manner. The high concentration of HF (1000 nM) upregulates chop but represses the induction of other ISR-related genes. This result corroborates previous findings from different species demonstrating that Chop overexpression contributes to a negative feedback loop responsible for attenuating the starvation-induced GCN2 response. They also demonstrated RT specificities for amino acid dependencies, time response, and the activation levels of their downstream targets [ 109 ]. They concluded that RT cell lines could be an alternative in vivo to analyze nutrition-related queries in Rainbow trout and other carnivorous fish using dietary proteins that provide most of energetic metabolism.

The regulations of atg4, lc3b, and sqstm1 observed in RTH-149 cells were previously described in a mouse cell line to be induced following starvation in a GCN2/ATF4-dependent manner [ 110 ]. The starvation-induced autophagy kinetics measured in RTH-149 cells matches with the cells of starved mouse embryonic fibroblast (MEF) [ 111 ]. That indicates the amino acid sensing and mTOR activation in RTH-149 cells follow the mechanisms shared between trout, human, and mice cell lines [ 112 , 113 ] and that has been conserved throughout evolution.

Several fish cell lines have been used as in vitro models to study elongation and desaturation of different PUFA. These in vitro models were also useful for unveiling the pro- inflammatory mechanisms underlying the relationship between dietary PUFA and cardiac lesions in salmon [ 46 ], the effect of fatty acid diet on fish inflammatory responses [ 122 , 123 ]. To study fish nutrition and metabolism, cell lines provide a great interest near future, especially advanced methods, such as CRISPR/cas9 and that may certainly work with new feed formulations for the development of sustainable aquaculture.

With global biodiversity rapidly declining, the need to preserve and conserve biological specimens becomes crucial. Cryopreservation techniques have long been used in agriculture for conservation. Proper freezing of cells can generate a bank of genetic material that can remain viable for hundreds or even thousands of years in the future, with the potential not just to act as reference specimens, but the capacity to regenerate live individuals of a species. While classic cryopreservation methods result in frozen sperm, which would need a fresh egg or frozen embryos–which poses challenges for proper freezing–new technology allows for the production of viable offspring from spermatogonial stem cells of fish.

The rapidly increasing number of fish cell lines raises the need for their long-term storage and conservation in different locations. Fish cell lines are not only the source material for in vitro research but also critical for the conservation of fish germplasm. Integrated efforts protect animal populations within their natural habitat (in situ conservation) and outside their natural environments ( ex-situ conservation). Similarly, ancillary conservation facilities like repositories of serum, DNA, and cell lines have been supporting basic and applied research [ 114 ]. Bio-banking is emerging as one of the most efficient approaches to provide security at the highest level against the loss of diversity of species [ 115 ]. Caulfield and Murdoch have critically reviewed various social and technical issues of biobank globally including public perception, biorights, privacy, technology, and commercialization [ 116 ]. The standard operating procedure should be followed for the long-term conservation of fish cell lines. The stability of the cell lines and recovery rate of the fish cell lines should be assessed using different replicates in the freezing medium at different passages to minimize the loss of cells. Working and master stocks of the fish cell lines also need to be maintained separately in the cell line repository. An automated controlled-rate programmable freezer would be ideal to provide reproducible cryopreservation with an optimized freezing program as per the cell’s requirement [ 117 ]. A simpler device like cryocan filled with liquid nitrogen may also be used for the storage of cell lines. Researchers can access fully characterized and quality-controlled cell lines from a repository without spending time to develop as per their requirement and at a minimal cost. The repository acts as an “insurance” to secure the loss of cell lines developed by a single laboratory. There should be many standby repositories at different places to avoid loss of the cell lines in case of any catastrophic event. Hence, the cell line repository facilitates promote the propagation of in vitro research as well as the conservation of fish germplasm.

The leading cell line repositories in the world like American Type Culture Collection (ATCC), European Collections of Cell Cultures (ECACC), German Collection of Microorganisms and Cell Cultures (DSMZ) have been providing characterized and authenticated cell line to researchers across the world. Details of cell lines maintained in the repositories worldwide are given in Table 2 . Some of the cell lines in the repository suffer from misidentification and contamination due to multiple transfers between laboratories. A certificate citing STR profile for each line is essential to guarantee authentic and contamination-free cell lines [ 12 ]. A very encouraging progress in the development of cell lines from different fish species including aquaculture species has been observed in India during the last decade. DBT, Govt. of India New Delhi, India has been instrumental in funding various projects in the development and characterization of fish cell lines in India which resulted in a rapid increase in the number of fish cell lines. This raises the need of establishing fish cell line repositories at a national level for the conservation of fish cell lines in secured places. The authors (M Goswami and W S Lakra) have developed a state-of-the-art facility for the development and storage of cell lines at ICAR-National Bureau of Fish Genetic Resources (NBFGR), Lucknow, India. Recognizing the expertise and research contributions of the authors and other colleagues in the country in the fish cell culture area, DBT funded a megaproject to establish a National Repository of Fish Cell lines (NRFC) at NBFGR, Lucknow. This National Repository of Fish Cell Lines (NRFC) has been in operation at NBFGR, Lucknow, since 2010 which is serving as a National Referral Centre of fish cell lines for research use in the country and abroad. More than 50 fish cell lines from 24 different fish species are being maintained and cryopreserved in the NRFC (Table 3 ). The facility provides services for deposition, characterization, cryopreservation, and distribution of fish cell lines to the scientific community in India. Many cell lines have been supplied to domestic researchers for their research experiments. This cell line repository would play a critical in contributing to the global biobank as many international scientific communities have expressed interest in sharing fish cell lines from India for collaborative in vitro research.

The growing interests in conducting in vitro research using fish cell lines have necessitated intensification of efforts to maintain constant quality and authenticate cell lines using standard protocol throughout their in vitro life. Fish cell culture has been increasingly used in modern biological research. However, fish cell culture research confronts many challenges like misidentification and contamination. As aquaculture continues to grow worldwide, the application of the fish cell lines in addressing fish disease, genetics, and biotechnological interventions will also increase many folds. Although the total number of fish cell lines has been increasing development and characterization of cell lines from crustaceans and other important marine and aquaculture species are still elusive. Preliminary efforts have been made for the development of stem cell cultures from fish but this area needs more focus to explore their use in modern aquaculture and biotechnology. The potentials of fish cell lines in developing vaccines for aquaculture and other derivable products from cells have yet not been explored fully. Hence, scaling up the fish cell culture systems is essential to grab the opportunities of using fish cell cultures in cell-based aquaculture .

There is a need for global networking and collaborations towards applications of fish cell lines for carrying out advanced in vitro research in fisheries and aqua, blue economy, human health, and environmental management. With technological interventions, fish cell lines could be explored to produce several new products. The information provided by the authors in this paper will add new knowledge to the global database of the fish cell lines besides their potential application in the advancement of aquaculture biotechnology and fisheries science research.

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Acknowledgements

Director, ICAR-National Bureau of Fish Genetic Resources, Lucknow; Director, ICAR-Central Institute of Fisheries Education, Mumbai are thankfully acknowledged for providing the facilities. The authors gratefully acknowledge the Department of Biotechnology, Govt. of India, New Delhi for financial support.

No funding was received to assist with the preparation of this manuscript.

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Authors and affiliations.

ICAR – Central Institute of Fisheries Education, Mumbai, 400061, India

M. Goswami & B. S. Yashwanth

Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, Canada

Vance Trudeau

NABARD Chair Unit, ICAR-Central Marine Fisheries Research Institute, Mumbai Research Centre, Versova, Mumbai, India

W. S. Lakra

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All the authors contributed to the preparation of this manuscript. MG, and YBS were responsible for the literature search and the first draft of this article. VT and WSL were responsible for language polishing and further editing the manuscript. All authors read and approved the final manuscript. Dr. MG Covered a major aspects of fish cell line for in vitro research like, the current status of fish cell line, novel characterization methods for fish cell line, biobanking, role of cell line in fish health management including the pathological and immunological studies, gene editing of fish cell line and the major contributor of the establishment of NRCF, India. YBS Contributed in the studies on vaccine and other products developed from fish cell culture and the studies on cell-based aquaculture. VT Contributed in the studies of fish cell line in transgenic studies and reproductive biology. WSL Contributed in the studies of fish cell line in toxicological research and environmental monitoring.

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Correspondence to M. Goswami .

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Goswami, M., Yashwanth, B.S., Trudeau, V. et al. Role and relevance of fish cell lines in advanced in vitro research. Mol Biol Rep 49 , 2393–2411 (2022). https://doi.org/10.1007/s11033-021-06997-4

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DOI : https://doi.org/10.1007/s11033-021-06997-4

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MCF-7 as a model for functional analysis of breast cancer risk variants

1 Center for Neurodegenerative Science, Van Andel Research Institute, Grand Rapids, MI, United States

Gerhard A Coetzee

Steven e pierce.

Authors’ contributions

Associated Data

Publicly available MCF-7 H3K27ac and CTCF ChIP-Seq, and HMEC RNA-Seq datasets used during the current study are available from the encyclopedia of DNA elements (ENCODE), [ https://www.encodeproject.org/ ]

All data generated during this study are included in this published article [and its supplementary information files ] or have been deposited in NCBI’s Gene Expression Omnibus ( 34 ) and are accessible through GEO Series accession number {"type":"entrez-geo","attrs":{"text":"GSE130852","term_id":"130852"}} GSE130852 .

Breast Cancer (BCa) genetic predisposition is governed by more than 142 loci as revealed by genome-wide association studies (GWAS). The functional contribution of these risk loci to BCa remains unclear and additional post-GWAS analyses are required.

We identified active regulatory elements (enhancers, promoters, and chromatin organizing elements) by histone H3K27 acetylation and CTCF occupancy and determined the enrichment of risk variants at these sites. We compared these results to previously published data and for other cell lines, including HMEC cells, and related these data to gene expression.

In terms of mapping accuracy and resolution, our data augments previous annotations of the MCF-7 epigenome. After intersection with GWAS risk variants we found 39 enhancers and 15 CTCF occupancy sites that, between them, overlapped 96 BCa credible risk variants at 42 loci. These risk enhancers likely regulate the expression of dozens of genes, which are enriched for GO categories including estrogen and prolactin signaling.

Conclusions

Ten (of 142) BCa risk loci likely function via enhancers which are active in MCF-7 and are well suited to targeted manipulation in this system. In contrast, risk loci cannot be mapped to specific CTCF binding sites and the genes linked to risk CTCF sites did not show functional enrichment. The identity of risk enhancers and their associated genes suggest that some risk may function during later processes in cancer progression.

Here we report how the ER+ cell line MCF-7 can be used to dissect risk mechanisms for BCa.

Introduction

According to the traditional model of carcinogenesis, normal tissue undergoes rounds of oncogenic mutations during proliferation that eventually leads to metastatic tumor growth. An individual’s inherited genetic predisposition for cancer, which can be measured by linkage analysis or genome-wide association studies (GWAS), influences the rate of those oncogenic somatic mutations and the environment in which the tumors develop ( 1 ). In breast cancer (BCa) rare but high-penetrance inherited mutations to genes, such as BRCA1 and BRCA2 , contribute about 30% to the familial risk of developing breast cancer and, in general, have well understood biological consequences ( 2 ). However, only about 5%−10% of BCa cases are actually associated with this type of germline mutation. In contrast, GWAS show that low-penetrance but common genetic variants explain up to 50% of disease heritability and contribute significant risk to the development of both familial and sporadic BCa ( 3 ). Unlike high-penetrance rare mutations, in most cases, these other risk genotypes are poorly understood and do not alter protein coding. Elucidating the functional basis of these common risk variants is therefore of great importance.

A recent GWAS uncovered 65 new breast cancer risk loci contributing to a total of roughly 142 reproducible breast cancer risk loci containing over 38,000 statistically significant (combined p.value < 5×10 −8 ) or near significant (combined p.value < 1×10 −5 ) risk variants ( 4 ). These variants primarily consist of single nucleotide polymorphisms (SNPs) (we use ‘variant’ and ‘SNP’ interchangeably in this text). Due to the large number of risk-associated SNPs as well as to their presence in noncoding DNA, it can be difficult to pinpoint which SNP or combination of SNPs are causal for a disease, let alone explain the biological mechanisms/genes involved. This will only become more challenging as the number of identified risk SNPs is expected to increase as the sizes of case-control studies grow larger in the near future. Our goal is therefore to prioritize SNPs based on their potential effects on cell type specific genomic activity.

Risk SNPs are non-randomly distributed throughout the genome and have been shown to be enriched in tissue-specific noncoding regulatory elements (REs), mainly in enhancers ( 5 – 7 ). Regulatory elements are defined as regions of non-coding DNA that regulate the transcription of genes. Enhancers are a class of regulatory element that influence cell fate and development through coordinated interaction between transcription factors (TFs) and their target promoters to alter the transcription of genes ( 8 ). Enhancers are marked by surrounding histone modifications and nucleosome depletion. The histone modification most often used is H3K27 acetylation, since it has been shown that it marks active (engaged) enhancers ( 9 ). Enhancers are also relatively transient, and their activity is dictated by extracellular signals that activate complex enhancer networks to promote cell type and condition specific gene expression. Common breast cancer risk variants present in gene REs are likely to subtly alter gene expression patterns, compared to protective alleles, in ways that predispose some individuals to cancer. For example, studies of the noncoding region 8q24 upstream of MYC have shown enrichment of variants associated with different tumor types, including breast, in multiple enhancers at this location ( 10 – 12 ).

Another important feature shown to coincide with risk SNPs is CTCF (CCCTC binding-factor). CTCF is a highly conserved 11-zinc finger protein and is the only known insulator protein in vertebrates ( 13 ). It is most often enriched at loop and topologically associated domain (TAD) boundaries that separate transcriptionally active and repressed genes ( 14 ). This implicates its importance in the organization of chromatin compartments to prevent aberrant gene-enhancer interactions. Lupianez et al. ( 15 ) showed that removing the CTCF associated boundary elements at the Epha4 TAD causes abnormal interactions between adjacent TADs and expression of genes within those TADs. Others have shown that CTCF is important for spatial organization of the genome for proper induction and silencing of transcription ( 14 , 16 ). Although the relationship between SNPs and CTCF is less clear than that of SNPs within enhancers, SNP mediated disruptions of CTCF consensus sequences has been shown to change the binding affinity of the CTCF protein ( 17 ). This could result in gene spatial network rewiring that has a strong influence on gene expression changes that indirectly lead to BCa. Such rewiring was demonstrated by a CRISPR-mediated deletion of prostate cancer risk-associated CTCF sites, which identified repressive chromatin loops [17].

In the present study, we used the MCF-7 cell line as a model of breast cancer. It is one of the most utilized breast cancer cell lines in cancer research due to its long history and expression of the estrogen receptor (ER) ( 18 ). The majority of patients (72.7%) with a known HR/HER status (estrogen receptor + progesterone receptor/human epidermal growth factor receptor) are HR+/HER2- ( 19 ). MCF-7 cells are one of the few breast cancer cell lines that is also HR+/HER2-, making it an extremely relevant model for the study of invasive breast cancer. Even, so there is currently a lack in the literature of post-GWAS analyses based on experimental manipulation in ER+ systems, including MCF-7 ( 20 ). Although a detailed meta-analysis of BCa risk loci was recently conducted and credible risk variants (CRVs; see methods for a detailed definition) genome-wide were defined ( 4 ), potential functionality was only alluded to in broad terms. Here, we analyze epidemiologically defined genetic risk loci within the MCF-7 cancer cell line. In so doing, we identified functional targets that can further be tested by genetic manipulation. We report a priority list of BCa risk enhancers and risk CTCF-binding-sites tailored to the MCF-7 cell line.

Materials and Methods

Cell culture:.

MCF-7 cells were obtained from ATCC and cultured in DMEM (ATCC, cat # 30–2003) supplemented with 10% FBS and 0.01 mg/ml human recombinant insulin (Gibco, ref # 12585–014). They were incubated in a humidified 37°C, 5% CO 2 incubator. For routine passaging, cells were grown in T25 and T75 culture flasks and passaged using 0.25% Trypsin/EDTA.

For ChIP we followed previously published protocols from Rhie et al ( 21 ) with slight modifications. Roughly 30–40 × 10 6 cells were used per ChIP. Upon reaching 70–80% confluency, cells were directly fixed in T75 flasks by adding 16% formaldehyde to the culture medium to a final concentration of 1%. The reaction was quenched for 5 minutes at room temperature with 10X (1.15 M) glycine. Using a Bioruptor Pico (Diagenode, Cat # B01060001) the isolated chromatin was sonicated for 30 second on and 30 second off cycles to yield DNA fragments between 200 and 500 base pairs. 100 ug of sonicated chromatin was used for immunoprecipitation and 1 ug (1%) was used for the input control. To probe for CTCF or H3K27ac, samples were incubated at 4 °C overnight with a primary antibody [CTCF: Cell Signaling monoclonal (D31H2), Cat# 3418; H3K27ac: Active Motif, Cat #39133] or an IgG control (Sigma, Cat # R9133). A/G magnetic beads (Pierce, Cat # 88802) were then incubated with the samples for 2 hours at 4 °C. Following this incubation, the beads were washed with a series of buffers of varying salt concentrations before overnight elution at 67 °C. The ChIP, IgG, and Input samples were all purified using a QIAprep Spin Miniprep Kit (Qiagen, Cat # 27104).

Construction and Sequencing of ChIP-Seq Libraries

Libraries for input and IP samples were prepared by the Van Andel Genomics Core from 10 ng of input material and all available IP material using the KAPA Hyper Prep Kit (v5.16) (Kapa Biosystems, Wilmington, MA USA). Prior to PCR amplification, end-repaired and Atailed DNA fragments were ligated to Bio Scientific NEXTflex Adapters (Bio Scientific, Austin, TX, USA). The quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Sequencing (75 bp, single end) was performed on an Illumina NextSeq 500 sequencer using a 75-bp sequencing kit (v2) (Illumina Inc., San Diego, CA, USA). Base calling used Illumina NextSeq Control Software (NCS) v2.0, and the output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.

Identification of ChIP-Seq Peaks

Two biological replicates of MCF-7 were used for input and ChIP for H3K27Ac and separately for CTCF. Following sequencing, fastq files were aligned to the HG19 genome assembly using default setting for BWA v0.7.15 ( 22 ). Mapped sequencing depth was 40 – 50 million reads ChIP, ~110 million reads input for CTCF and 60–70 million reads ChIP, ~150 million reads input for H3K27ac. Aligned reads were called using MACS2 v2.1 at a liberal FDR cutoff of 0.1 ( 23 ). For CTCF comparison to ENCODE data (ENCODE1 = ENCSR000DMV, ENCODE2 = ENCSR560BUE, primary antibody: Millipore polyclonal (07–729); ENCODE3 = ENCSR000DWH primary antibody: Cell Signaling (2899)), analysis started with fastq files and were aligned with BWA as above, except: reads less than 50 bp in our data and reads less than 20 bp in the ENCODE data were removed using Trim Galore prior to peak calling. Peaks were filtered by IDR (V2.0.2) according to default settings. For H3K27Ac, Trim Galore was not used and ENCODE comparison was done starting with peaks files (also generated using BWA and MACS2). Existing data for MCF-7 H3K27ac ChIP-Seq ENCODE1 (ENCSR000EWR, read length: 32 bp, ChIP depth = ~20 million reads, input = ~10 million reads), ENCODE2 (ENCSR752UOD, read length: 36 bp, ChIP depth: rep1, 60 million and rep2, 20 million reads, input: 70 – 80 million reads), and for HMEC (ENCSR000ALW) were used for comparison. NarrowPeak calls (i.e. ENCFF187RUK, ENCFF37ORFF, ENCFF537JMI, and ENCFF208IPB) were filtered by IDR to generate plots in Figure 1 and for peak coordinates. All H3K27Ac peak data was filtered by IDR (V2.0.2) with the following parameters for peak-merge: --rank p.value --soft-idr-threshold .01 --peak-merge-method max ( 24 ). FRiP was calculated using deepTools (V.3.1.3) ( 25 ). Intersected datasets are defined by coverage, not peaks, i.e. every bp annotated in all datasets is included; overlapping peaks are not merged. Prediction for allele dependent CTCF binding was made using MotifBreakR (V.1.8) ( 26 ).

BCa Risk SNPs

All SNPs used here derived from the recent BCa GWAS meta-analysis by Michailidou, et. al. ( 4 ). Their report provided a set of 11.8 million 1000 Genomes SNPs which were associated with BCa and 20,989 SNPs with significant or near significant (combined p < 1 × 10 −5 ) association values. Across 142 regions they selected the most significant SNP or SNPs and identified those nearby SNPs within 500 Kb and 2 orders of magnitude significance. These they called credible risk variants (CRVs). The risk loci correspond roughly to sets of significant risk SNPs (combined p. value < 5×10 −8 ) with at least 400 to 500 Kb intervening distance. We combined the sets of CRVs and associated annotations from their Supplemental Tables: 2, 6, 8, 11, 13, and 14 to produce a list of 4,453 BCa CRVs corresponding to 65 new risk loci and 77 re-confirmed previously published risk loci. Across these regions, we also selected the most significant SNPs per locus and used RaggR ( 27 ) to obtain the set of 8,687 phase 3 1000 genomes in LD R 2 > 0.8 based on European linkage maps.

Overlap and Enrichment of BCa Risk SNPs in Regulatory Elements

SNP enrichment was obtained using Bedtools v2.26.0 to overlap risk SNP genomic locations (hg19) with RE locations ( 28 ). The ratios of overlapping risk SNP/total risk SNPs were compared to the background ratio: either based on of the overlap of all 11.8 million background SNPs with association values by Michailidou, et. al. ( 4 ) or based on the ratio of only those 11.8 million background SNPs within 1Mb of each risk locus (1.25 million SNPs). The hypergeometric distribution was used to obtain significance values of overall overlap frequencies ( Sup. Table 2 ).

These enrichment ratios are depicted in Figure 2B–D and are based on the overlap of all BCa risk variants with all enhancers, compared to a single background set. This is useful for determining in general whether the entire set of risk SNPs is related to a specific type of genomic element or to tissue-specific activity. The metric quantifies how relevant any particular model is likely to be for examining risk. Unfortunately, when examining each locus individually, this sort of enrichment calculation can be misleading. This is because, while the background set of SNPs are independent at both the level of genome and single locus, due to genetic linkage, risk SNPs are not independent of each other at the level of a locus. Although multiple significant risk SNPs can be present at a single locus, they should not be treated as independent, but instead represent a single risk signal. As such, an enrichment score for a single locus can be a function of the local LD structure, which we do not expect to be a good indicator of disease relevance. For this reason, the background rate of overlap should be adjusted separately for each locus.

To compare the significance of risk span/RE overlap at each locus separately, the most distant CRVs for each locus were used to define a CRV span and the proportion of that span, in bp, which overlapped RE peak coverage was calculated using Bedtools. That value was then ranked against a background distribution of overlap proportions for each of the 142 loci. The background distributions were calculated by permutation testing using R, wherein background SNPs within 1 MB were randomly drawn 10,000 times and used to form the center of a span equal in length to the risk span, and then overlapped with REs. This comparison generates a probability value corresponding to the proportion of the background distribution with an equal or greater amount of overlap as the risk span. The same set of random spans for each locus were used to compare overlap with different tissue or types of REs.

These comparisons were used to generate the heatmap shown in Figure 3. This was done, in addition to using more common enrichment scores, primarily because we think that the location of individual risk variants within a locus is affected by how risk association is propagated during imputation according to LD related to some single risk element. Thus, these multiple variants represent a single signal. Enrichment calculations, and in particular, statistical tests which assume multiple independent tests are not valid. Randomly drawing individual SNPs from a large set of background SNPs for comparison is likewise not appropriate. Instead we asked: given a span of DNA associated with breast cancer risk, what percentage overlaps an MCF-7 RE and how does that compare to a background of equal sized spans nearby? We believe that the identification of likely risk region is probably more accurate for small, well defined REs in inactive regions, than for large REs in regions of high activity. For instance, Supplemental Figure 2 shows 2 loci with high risk SNP enrichment but with widely spaced CRVs so that they are characterized by a low span overlap significance and correspond to ambiguity in risk RE identification.

Construction and Sequencing of Directional mRNA-Seq Libraries

Libraries were prepared by the Van Andel Research Institute Genomics Core from 1 μg of material using the KAPA Stranded mRNAseq Kit (v4.16) (Kapa Biosystems, Wilmington, MA USA). RNA was sheared to 250–300 bp. Prior to PCR amplification, cDNA fragments were ligated to Bio Scientific NEXTflex Adapters (Bioo Scientific, Austin, TX, USA). The quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems).

Differential Gene Expression Analysis

Existing RNA-seq data from ENCODE for MCF-7 (ENCSR000CPT) and HMEC (ENCFF000GDZ) and from NCBI for HMEC ( {"type":"entrez-geo","attrs":{"text":"GSE47933","term_id":"47933"}} GSE47933 ) were used for comparison, without alteration. To these we compared newly generated expression data from 8 WT biological replicates of MCF-7, which were sequenced in two separate experiments. For our experiments 8 different MCF-7 WT clones were expanded until reaching 80% confluency in a T25 flask. RNA was isolated using a Qiagen RNeasy mini kit (Cat # 74104). Paired-end mRNA libraries were then prepared by the Van Andel Research Institute Genomics Core as described in the previous section. Following sequencing, fastq files were aligned to HG19 using STAR v2.5 ( 29 ). Alignments (bam files) were converted to feature counts using HTSeq v0.6.0 referenced against the ENSEMBLE annotation of HG19: Homo_- sapiens.GRCh37.87.gtf counting against the feature “exon”, grouped by “gene_id”, and using the strand parameter “reverse”. This set included exon locations for 57,905 genomic entities including pseudogenes, lncRNAs, and 20,356 protein coding genes. The resulting gene_id map counts were normalized using edgeR (TMM) and tested for significant differential expression with Limma and Voom, in R (v3.3.1) ( 30 – 32 ). The normalized count data for all 8 datasets revealed, through principle component analysis (PCA), that there was very high similarity between our WT MCF-7 which was distinct from the encode data ( Fig. S3 ). In total 57,905 genes were mapped, however in order to combine our data with existing MCF-7 and HMEC RNA-seq data gene_ids, which were not reported in the published studies, were filtered out.

Gene Ontology Analysis

Gene ontology enrichment analysis was done using String v.10.5 ( 33 ).

Availability of Data and Material

Mcf-7 chip-seq and rna-seq analyses.

With the goal of identifying which genetic BCa risk variants are functional in the MCF-7 cell line, we first attempted to locate REs that are active in MCF-7 cells using Chromatin Immunoprecipitation followed by high-throughput sequencing (ChIP-Seq). ChIP-Seq has become widely-used in many cell types, including MCF-7 to map genome wide histone modifications and transcription factor binding sites. Whereas this method has been integral in locating gene REs, it can be highly variable ( 35 ). Antibody quality, sample preparation, sequencing depth, and MCF-7 cell line heterogeneity are the main sources of technical variability. Furthermore, following successful immunoprecipitation and sequencing, various algorithms then must be employed to define peaks: regions showing a high number of mapped reads relative to background. In particular, sufficiently deep sequencing is important for this part of the process wherein active REs are defined. Deeper sequencing allows for a lower relative detection threshold and so can capture low-expression transcripts, but also generates greater discrimination among peaks and more signal relative to control and so can map features with higher resolution and more reproducibly. This is important for the detection of variable regions that may not have robust enrichment but are still highly active and functionally important ( 35 , 36 ). An example at one locus showing 3 different H3K27ac CHIP-Seq experiments, including our own reported here, is shown in Figure 1a.

In addition to technical variation, MCF-7 is prone to instability and major genetic and expression changes can be present between subclones ( 18 ). Therefore, prior to any genetic manipulation of MCF-7 for disease modeling, recent and accurate characterization is recommended. We reviewed existing ENCODE MCF-7 CHIP-Seq H3K27ac and CTCF datasets and compared these studies to our own ( Figure 1B – D ). We mapped a total of 14,722 H3K27ac peaks, corresponding to a coverage of roughly 35.2 Mb of DNA ( Sup. Table 1 ). In comparison, the Encode 1 and Encode 2 datasets generate 11,304 and 20,102 reproducible peaks covering about 13.3 and 49.4 Mb, respectively ( Figure 1 ). However, peak size can vary, even if the same peaks has been called in multiple datasets. By comparing our peak locations (labeled ‘Coetzee’) to the ENCODE datasets, we found that only 37% of our peaks overlapped H3K27ac regions from both ENCODE datasets (by at least 1bp). In addition, 21% are completely unique to our data, 1% overlap peaks in Encode dataset 1 (but not 2), and 41% overlap peaks present in Encode dataset 2 (but not 1). Examining unique DNA coverage across the three datasets yields a total of 61.4 Mb, which are annotated as H3K27ac in at least 1 experiment and 9.7 Mb (15%), which are annotated in all three datasets ( Figure 1E ). Based upon an analysis of the proportion of reads in peaks (FRiP), which indicates the specificity of library following immunoprecipitation, our data is similar to that of ENCODE 2, both studies of which used the same primary antibody for H3K27Ac [ Sup. Figure 1 ]. The Encode 1 dataset in contrast (the oldest study) used a different antibody and shows a lower proportion of reads in peaks. Analyzing the reproducibility of peak calling between the 3 studies showed larger differences, as seen by irreproducible discovery rate (IDR) analysis. This analysis filters out peaks which are not similar across two biological replicates. In this case our data retained 62% of defined peaks, ENCODE 2 retained 44%, and the oldest data, ENCODE 1 retained only 23% of peaks. ENCODE 1 used both a different primary antibody and was sequenced to a much lower depth for both input and control.

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A) Genome browser view of representative locus near ESR1 . B) IDR plot showing the correspondence between replicates 1 and 2 and the threshold for filtering (in black < 0.01) for our H3K27Ac ChIP-Seq (Coetzee) C) for Encode dataset 2, and D) for ENCODE dataset 1. E) Comparison of H3K27Ac ChIP-Seq annotation coverage. F) Proportion of IDR defined H3K27Ac peaks from the our (Coetzee) data that overlap peaks in ENCODE1, ENCODE2, both, or neither; by at least 1 bp. G) Comparison of CTCF ChIP-Seq annotation coverage. H) Proportion of IDR defined CTCF peaks from the our (Coetzee) data that overlap peaks in ENCODE CTCF 2, ENCODE CTCF 3, both, or neither; by at least 1 bp.

We used the same approach in comparing our MCF-7 CTCF data with three separate experiments from ENCODE, (labeled 1–3 and unrelated to the H3K27Ac ENCODE data). We detected almost double the number of peaks in our data set (65,997 peaks) compared to the other three ENCODE datasets with 38,553, 33,312, and 36,057 peaks respectively, that pass an IDR cutoff of q < 0.01 (IDR score > 830) ( Sup. Table 1 ). When combining all three datasets, they only share 20,601 common peaks. After removing CTCF ENCODE1, the least reproducible data, we further evaluated CTCF ENCODE datasets 2 and 3 in comparison to our own. With our CTCF ChIP-Seq data we detected a substantially greater amount of unique CTCF bound DNA (20.5 Mb) across the genome in comparison to CTCF ENCODE 2 and 3 (0.5 and 0.03 MB) ( Figure 1G ). The majority of CTCF sites detected by the CTCF ENCODE 2 and 3 datasets were also detected in our data. Out of the 65,997 CTCF peaks found in our experiment, 39% were unique to our dataset, 10% were shared with CTCF ENCODE 3 but not CTCF ENCODE 2, 7% were shared with CTCF ENCODE 2 but not CTCF ENCODE 3, and 44% were shared between all three datasets ( Figure 1H ). These data illustrate that CTCF peaks are largely conserved across datasets, but deeper sequencing may improve the detection of weaker CTCF binding signals.

Coincidence of BCa Risk Variants with Regulatory Regions

Although most risk variants do not alter protein coding, they must still show allelic differences in some functional aspect of genome biology if they are causally related to the disease. Identifying active regulatory regions that coincide with risk variant locations can point to such mechanisms. In order to identify BCa risk processes which are active in MCF-7, we intersected the locations of risk variants with both our own and the ENCODE MCF-7 ChIP-Seq H3K27ac peaks and CTCF binding peaks. To maximize both sensitivity and specificity multiple sets of SNPs were used in this analysis based on different definitions of which variants are most likely to confer BCa risk, all deriving from Michailidou, et. al. ( 4 ) and based on different thresholds for significance (see Methods for details, Sup. Table 2 ). Overall, we examined GWAS BCa risk variants corresponding to 142 separate loci. These loci each had one or small number of equally most significant variants per locus, with 210 in total. Additionally, Michailidou, et. al. ( 4 ) reported a set of credible risk variants (CRVs) for each locus, with an average of 31 (stdev. 50) CRVs per locus that spanned an average of 114 Kb (stdev. 179 Kb). CRVs are most likely to be functional and are based on a locus specific significance threshold (see methods ).

By examining the location of genomic regulatory activity with respect to risk variants, we can most easily infer which risk loci are not functional within a particular cell line. In MCF-7, 76 (of 142) breast cancer risk loci, and 93% of total reported CRVs, do not overlap any active REs. It is therefore very unlikely that these 76 loci confer risk via processes which are active in MCF-7. In contrast, the remaining 66 risk loci may function via processes that are intrinsic to, and active in, some breast cancer cells, making them logical targets for experimental manipulated in MCF-7. However, the possibility of spurious coincidence remains, as some rSNPs will overlap active REs simply by chance. Out of the 4,453 reported BCa CRVs only 70 (1%) CRVs overlap ( 31 ) H3K27ac peak locations present in all three datasets, while 191 CRVs overlap (80) H3k27ac peaks based on our data alone ( Sup. Table 2 ). Similarly, only 13 CRVs overlap a CTCF binding peak present in all of the MCF-7 CTCF ChIP-seq datasets, and 96 CRVs overlap (70) peaks based on our data alone. For CTCF, of the 13, just 5 (rs3008455, rs8103622, rs1800437, rs10231350, rs10116233), (and of the 96: 16 ( Sup. Table 4 )) are predicted to also cause allele dependent disruption of a known CTCF binding motif.

Coincidence of BCa Risk Variants with Regulatory Elements in MCF-7

If GWAS-measured BCa risk is functional in MCF-7 cells than we predict that the location of BCa risk SNPs is correlated with the locations of active MCF-7 enhancers and/or CTCF binding sites. That is, we expect that the character of MCF-7 as breast derived cancer cells can be captured uniquely by the location of regulatory elements and this unique activity pattern will predict the location of BCa CRVs. To test this hypothesis, we compared the location of BCa risk SNPs to entire set of imputed SNPs generated by Michailidou, et. al., the vast majority of which are not statistically associated with BCa. Polymorphisms are distributed non-randomly throughout the genome and tend to occur near active regions of transcription and so some BCa-unrelated SNP set is required to define a background rate of coincidence. The simplest calculation for SNP enrichment is to compare two ratios: the proportion of risk variants overlapping enhancers or CTCF sites and the proportion background SNPs overlapping the same. We made these enrichment calculations using slightly different definitions of MCF-7 enhancer or CTCF peaks, based on merging our data with the ENCODE data, and for different definitions of risk SNPs, as described above.

To calculate enrichment of BCa risk in MCF-7 then, we first measured the ratios of CTCF peaks overlap by both risk variants and with the entire set of imputed SNPs. By using this comparison, we found that overall enrichment of SNPs coinciding with CTCF binding sites is very modest and is close the overlap expected by chance ( Figure 2D ). Though the difference in enrichment scores between datasets was not large, CRVs showed the strongest enrichment for all ChIP datasets, indicating that this definition of risk SNPs may be the most relevant to BCa (see Sup. Table 2 for CTCF sites that overlap CRVs). Out of the 210 most significant risk variants dataset, our ChIP data were the only ones to coincide with any of them (rs4971059, rs56069439, rs12449271, and rs35383942). The effects of a CTCF motif disruption may apply across a broader range of cell types as CTCF occupancy is highly consistent ( 37 , 38 ). To demonstrate this in the scope of our work with BCa, we compared the location of HMEC CTCF peaks (from ENCODE) with our CTCF peaks and found that 99% (15,095/15,138) of HMEC peaks were shared (at least 1 base pair) with our MCF-7 CTCF peaks.

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A) Genome browser view of representative locus near ESR1 , showing in red: the location of risk SNPs and background SNPs, and in orange: the location of H3K27ac peaks, based on our data (Coetzee), our data intersected with ENCODE 2 (Intersect 2), our data intersected with both ENCODE data sets (Intersect 3), or the union of the 3 datasets. B) Heat map showing risk the SNP enrichment ratio in each of the 4 MCF-7 datasets of MCF-7 H3K27ac peaks, or in HMEC (which has no H3K27ac at the ESR1 locus) for each of the sets of BCa risk SNPs. C) Heat map showing the SNP enrichment ratio for enhancers that are unique to MCF-7, for MCF-7 enhancers that overlap HMEC enhancers, HMEC enhancers that overlap MCF-7 enhancers, or enhancers unique to HMEC. D) Heat map showing risk the SNP enrichment ratio for CTCF peaks.

In contrast to CTCF, we found highly significant enrichment of BCa risk SNPs in H3k27ac peaks (enhancers and promoters) for all sets of MCF-7 enhancers and all definitions of BCa risk variants ( Figure 2B ). For example, we found that BCa CRVs overlapped 81 MCF-7 H3K27ac peaks (based on our data alone) and were 2.3-fold more likely to coincide with peaks than were the background SNPs overall. This enrichment means that the location of MCF-7 enhancer activity is predictive of risk SNP location. Therefore, as expected, at least some gene regulation that is active in MCF-7 also increases the risk of developing BCa.

To begin to define risk element functionality, we first categorized MCF-7 H3K27ac peaks as enhancer or promoter based on gene transcription start site locations. As expected, promoter peaks were uniformly less likely than enhancer peaks to show enrichment for risk variants compared to background. Focusing on enhancers, we then compared MCF-7 active enhancer locations to normal human mammary epithelial cell (HMEC) active enhancer locations, based on ENCODE H3K27ac CHIP-Seq data ( Sup. Table 1 ). We reasoned that HMEC exhibits a noncancerous phenotype and so represents the active-enhancer profile of normal tissue, whereas MCF-7 cells represent the active enhancer profile of ER+ cancerous breast epithelial tissue. Therefore, they can be used to categorize risk enhancers active in two distinct cell types representing two extremes of breast epithelial cells. Although not yet demonstrated, we speculate that risk enhancers that are present only in HMEC point to risk imposed through pre-cancer cell functions (such as apoptotic checkpoints) that must be altered or bypassed for carcinogenic initiation. Risk enhancers present only in MCF-7 could affect mechanisms involved in tumor progression and malignancy (such as cell adhesion and immune avoidance). Finally, risk enhancers present in both cell types may regulate processes involved in BCa at all stages of oncogenic promotion (such as DNA synthesis and cell proliferation). The data presented in Figure 2C demonstrate that while all enhancer subtypes are enriched for BCa associated SNPs compared to background, MCF-7-only enhancers are most correlated with risk SNPs. This indicates that a greater proportion of MCF-7 specific processes are involved in conferring BCa risk compared to HMEC. It also suggests that some of GWAS measured risk is involved with later stage processes of cancer development.

Next, we measured the significance of overlap between risk SNPs and MCF-7 enhancers at each locus individually. The total number and relative locations of risk SNPs in a locus is influenced by both the underlying disease biology as well as the LD structure. Therefore, we sought to control for LD and simultaneously treat the rSNPs at each locus as fundamentally connected (see methods for more details). As a very simple partial solution to this problem we chose to merge the set of risk SNPs within a locus into a single peak or span. We then used permutation testing to compare that risk span (the distance between the outer most significant CRVs at a locus) against a background set of randomly sampled equal sized spans across the same locus (e.g. Figure 2A ). We measured the proportion of the risk span that overlaps active REs and generated a percentile score based on the number of background comparisons with greater overlap (Figure 3, Sup. Figure 2 , Sup. Table 3 ). A low value indicates greater confidence in the identification of a specific risk RE within a locus. This span overlap metric is helpful, in addition to SNP enrichment, and p-value ranking, in order to determine which REs are most suitable for follow up testing.

We calculated the risk-span-overlap at each locus for enhancers active in 11 related roadmap tissues as well as HMEC and MCF-7 ( figure 3 ). Notably, the largest group of loci show low overlap in any tissue. The remaining loci are easier to link to likely REs. For instance, at locus 6:151952332 near ESR1 , the actual BCa CRV risk span overlaps a greater proportion of MCF-7 enhancers than more than 98% of other possible cases within 1Mb. In contrast, no other cell types examined showed significant overlap. It is likely that the enhancer(s) at this locus, which completely overlaps BCa credible risk SNPs, is enhancing ESR1 expression. In the other cell types, no enhancer is present and ESR1 expression is probably off, such as it is in HMEC cells. BCa risk may be imposed through altered function of this enhancer, and this can be experimentally verified in MCF-7. Thirteen other loci show a greater overlap of risk SNPs with MCF-7 enhancers than expected by chance. Unlike ESR1 , the risk locus, 19:13954571, near NANOS3 , shows greater than expected overlap with MCF-7 enhancers as well as those of multiple other tissues. NANOS3 is expressed in many tissues and is likely involved in general proliferation. The function of this locus, then, may be related to and can be queried in MCF-7 in addition to the other cell types.

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ChIP-Seq data and Roadmap segmentations (subset for enhancers) were used to examine the significance of overlap with CRV risk spans for each of the 142 BCa risk loci (columns). Significance levels are depicted in red with darker red indicating a greater risk-span/RE overlap proportion compared to background levels for that locus. Dendrograms are based on complete linkage using city-block distance measurements of −log(p.value). The individual loci showing the most significant overlap are identified by chromosome and position underneath. These are the loci with the most specific correspondence between tissue-specific regulatory elements and BCa CRVs.

The risk span overlap metric may also be misleading in some cases. For example, widely spaced CRVs can give spans that overlap large amounts of RE even when no individual risk SNPs do. However, using this measure in conjunction with a simple enrichment calculation and also with GWAS significance-based SNP ranking is an improvement over any one method alone. Table 1 shows 10 BCa loci that are highly appropriate for study in MCF-7 based on all three metrics.

Listed are the 10 best BCa risk loci in MCF-7 and the corresponding 10 MCF-7 risk enhancers which are most suitable for follow up testing. Each enhancer overlaps the most significant GWAS SNP at the locus, and the locus shows both an enrichment greater than 2 (frequency of BCa credible risk variants (CRVs) overlapping enhancers compared to background overlap frequency), as well as a proportion of the CRV risk span that overlaps enhancers greater than more than 95% background overlap proportions. The enrichment and overlap values may be modified by overlap of risk SNPs with other enhancers at the same locus. The overall GWAS significance, ER+ GWAS significance, and reported genes from Michailidou et. al. are listed. Additionally, all but the enhancer at locus 19 are unique to MCF-7 and do not overlap HMEC enhancers locations, so the differentially expressed genes (relative to HMEC) within 500 Kb or 1 MB of each lead SNP are identified as putative risk genes in MCF-7.

CRVs Enh. Overlap	CRVs in Locus	Hyper. P.Val. (−log)	Ratio Enrich.	Span Overlap Signif. (−log)	Comb. GWAS P.Val.	Comb. GWAS P. Val., ER	Risk Enhancer Start	Risk Enhancer Stop	Gene	MCF-7 DE Genes 0–500Kb
8	50	4.29	5.79	1.72	2.38E-20	1.10E-07	10595877	10599012
1	22	0.65	3.31	1.71	9.07E-19	1.20E-15	4727872	4732087
10	10	12.50	17.64	2.19	2.84E-54	1.70E-22	151948050	151959756
2	8	1.50	6.62	1.66	5.61E-09	9.90E-09	102478133	102481107
30	42	23.50	8.93	2.15	1.73E-10	6.00E-08	9074392	9089320		NA
2	26	0.80	2.32	1.54	3.79E-08	1.40E-06	120831824	120833249
8	17	9.69	26.12	2.66	4.06E-27	2.90E-21	80647147	80651103
3	13	2.56	9.98	1.90	1.14E-10	2.40E-06	77769769	77772332	NA
19	43	13.35	8.74	2.74	1.08E-08	1.70E-05	13948902	13955856
2	2	4.32	143.22	2.52	1.87E-32	4.20E-23	16570278	16574820

Putative MCF-7 BCa-Risk Genes

In order to begin to characterize the processes active in MCF-7 that confer risk for BCa via GWAS-identified risk variants we sought to link risk enhancers to the genes which they may regulate. Therefore, we performed RNA-seq on MCF-7 cells and also compared that expression data to existing RNA-Seq data for both MCF-7 and HMEC cells ( Sup. Figure 3 ). Our data corresponded well to published MCF-7 data ( Sup. Figure 3a ). Combining the MCF-7 datasets, we found 11,309 protein coding genes expressed at an average of at least 1 count per million (CPM), whereas 12,294 genes were expressed in HMEC ( Sup. Table 5 ). Comparing the two cell lines, 10,657 genes (82.3%) were minimally expressed in both, and 8,087 genes were differentially expressed (adjusted p.value < 0.05 and fold change > 2) with 4,899 more highly expressed in HMEC and 3,188 more in MCF-7 cells. The expression changes between the cell lines were not unexpected. MCF-7 genes were, roughly, over-enriched for differentiated cell anatomical functions, migration, and intercellular interactions, and also enriched for KEGG cancer pathways. The HMEC genes were, roughly, over-enriched for proliferation, metabolism, and nuclear organization. For instance, the top 1000 genes most significantly upregulated in MCF-7 were statistically enriched for 382 GO biological processes including “vasculature development”, “anatomical structure morphogenesis”, “regulation of cellular component movement”, and “extracellular matrix organization” (FDRs = 5.2E-12, 5.2E-12, 2.0E-11, 2.2E-11) ( Sup. Table 5 . The top 1000 most significant HMEC genes were enriched for 63 processes including “RNA metabolic process”, “nucleic acid-templated transcription”, “nucleobase-containing compound metabolic process”, “RNA biosynthetic process” (FDRs = 7.0E-8, 7.0E-8, 2.1E-7, 2.2E-7) ( Sup. Table 5 ).

Multiple methods exist that attempt to pair enhancers with target genes ( 39 – 41 ). The simplest method, which we use here, is to identify nearby genes which are co-expressed with active enhancers specifically in a particular cell type, such as MCF-7. For this purpose, we used only one definition of MCF-7 enhancers based on our own H3K27ac data (see Figure 1 ) following the removal of promoter peaks. The majority of previously reported enhancer-gene regulatory pairing occur at distances less than 1Mb, though far cis and even inter-chromosomal interactions do exist ( 42 , 43 ). Based on our previous work we used here a distance threshold of 500 kb distance to identify putative risk genes expressed in MCF-7. By this proximity cutoff, 522 genes are near risk enhancers in MCF-7 cells. These potential risk-associated genes expressed in MCF-7 are statistically enriched for 102 GO biological process terms including “nucleosome assembly”, “DNA methylation”, “chromatin silencing”, and “positive regulation of DNA repair” (FDR = 1.4E-5, 7.9E-4, 9.6E-3, 1.5E-2) ( Sup. Table 6 ). Likewise, there are 386 putative risk genes expressed in HMEC and 260 genes common to both the HMEC and MCF-7 sets. HMEC were statistically enriched for 42 biological processes. However, almost no significantly enriched HMEC processes were unique, 80% were also significant for the set of MCF-7 risk genes. The exception being enrichment for GO pathways related to Notch signaling or apoptosis ( Sup. Table 6 ). This suggests that few BCa risk processes are unique to HMEC, compared to MCF-7.

A potentially more accurate method to link REs with target genes is by using expression quantitative trait loci (eQTL) data, from sources such as GTEx. These results are biased by tissue sample type and population origin and cannot identify changes is high variable or lowly expressed genes. However, an eQTL describes a direct association between the allelic variation at a SNP and an expression change for a gene within 1Mb. In order to integrate eQTL data we identified all SNPs (not just BCa risk SNPs) located within MCF-7 risk enhancers and queried the GTEx database for all significant eQTL genes, associated with those SNPs in breast tissue. We then removed from this set those genes which are not expressed in MCF-7. Doing so produced 48 genes ( Sup. Table 7 ). These genes were not enriched for any GO annotations and may be too stringently filtered to include likely risk genes. For instance, ESR1 , a gene known to be important in BCa, and near a risk enhancer ( Figure 1 ) has no identified significant eQTLs in the GTEx dataset. Moreover, the CRVs located at that risk locus, one of the most significant of all loci, have also not been measured to be eQTLs for any gene. Thus, this clear test case fails reidentification by GTEx. So, while eQTL can provide strong support for linking a risk locus to a gene, we think it is currently too restrictive for further use here.

Because enhancer regions active in MCF-7, and not active in HMEC, were most highly correlated with the locations of risk variants we sought to associate this subset of enhancers with putative genes. Enhancers alter the expression of nearby genes so we can assume that most genes that are regulated by this subset of risk enhancers will appear differentially expressed relative to HMEC expression levels, in which the risk enhancers are not active. Although most enhancers upregulate nearby genes, H3K27ac ChIP-Seq will also identify regulatory regions that can downregulate nearby genes. For this reason, we considered DE genes that were both up and downregulated with respect to HMEC. Based on 500 KB proximity, 138 DE genes are associated with MCF-7-only risk enhancers ( Sup. Table 7 ). These are enriched for multiple broad GO categories, but also including the KEGG pathways for estrogen and prolactin signaling, identified via 6 and 5 genes, respectively ( Sup. Table 7 ).

Finally, in table 1 we list risk MCF-7 enhancers at 10 BCa risk loci, which are highly enriched for SNPs, coincide with the span of DNA containing BCa CRVs above background levels, and show overlap for the most significant risk SNP. We linked MCF-7 genes to these 10 enhancers based on expression in MCF-7, significant difference in expression relative to HMEC, and a distance closer than 1 Mb. These represent the most promising enhancer targets for further functional analysis.

MCF-7 is a highly utilized breast cancer cell line. However, the range of its potential use for the dissection BCa GWAS risk is not immediately obvious. Based on the classical theory of carcinogenesis, cancer arises from normal tissue and proceeds through the stages of initiation, promotion, and progression. Genetic factors affecting any of these stages may be picked up in GWAS studies, but only a subset of these risk mechanisms are likely to be active in MCF-7 itself. Only those processes active in MCF-7 can in turn be easily manipulated. By comparing MCF-7 to HMEC we believe that risk arising from gene regulation involved in all three stages is active in MCF-7, but that MCF-7 is most suited for studying the risk mechanisms exacerbating tumor progression. In particular, estrogen and prolactin signaling gene networks are especially enriched in BCa GWAS risk biology in MCF-7. Although MCF-7 cells are classified as luminal A/ER+, it is worth noting that this cell line may still be relevant for tumorigenic processes in other subtypes including ER- breast cancer. The developmental lineage of breast tumor subtypes is complex and not fully understood. Based on the hypothesis that different subtypes may be derived from the same cell type of origin, some of the active enhancer-driven processes leading to cancer in each subtype are most likely not mutually exclusive. Further studies need to be done to evaluate the overlapping risk in multiple breast cancer cell lines of different classifications.

We found that the work reported here is more reproducible in defining MCF-7 specific H3K27ac histone marks and CTCF occupancy than that in previously reported ENCODE datasets. The discrepancy in the total amount of H3K27ac or CTCF DNA and in continuously designated regions (peaks) is likely due both to differences in sample preparation and sequencing depth as well as to intrinsic differences between subclones.

A potential next step in dissecting BCa risk in MCF-7 is perform allelic replacement or to delete the entire risk regulatory elements using CRISPR-CAS9 gene disruption. We found that multiple enhancers are well suited for follow-up experiments and a smaller number of CTCF sites may also be suitable. The CTCF protein function is well characterized and compared to enhancers, for which multiple unknown transcription factors may be acting, is fairly simple. Moreover, allele dependent CTCF binding can potential disrupt large TAD regions leading to large expression changes to nearby genes. For these reasons CTCF can be an ideal target. Unfortunately, we found very low enrichment of rSNPs, implying more BCa risk loci function via enhancers in MCF-7. It is possible that the enrichment scores maybe less informative for CTCF, though. CTCF binding sites/peaks are much more specific and narrower than that of H3K27ac peaks and are less likely to overlap multiple risk SNPs simply due to the close proximity of risk SNPs to each other. This and the greater number of CTCF peaks (which are largely tissue invariant) may contribute to lower overall enrichment. In total we found 5 loci which are good targets for further CTCF examination.

In contrast, at least 10 loci point to specific enhancers as casual in BCa risk. For enhancers we found the greatest degree of enrichment of risk variants in the MCF-7 cell line as compared to a normal precursor model (HMEC), indicating that MCF-7 cells are highly relevant for the study of processes leading to abnormal cell growth and tumor formation.

Overall, our results reported here bring into focus how MCF-7 can be used as a model to reveal BCa risk mechanisms in ER-positive genetic predisposition. For enhancers we found the greatest degree of enrichment of risk variants in the MCF-7 cell line as compared to a normal precursor model (HMEC), indicating that MCF-7 cells are highly relevant for the study of processes leading to abnormal cell growth and tumor formation.

Supplementary Material

Acknowledgements.

We would like to thank everyone who contributed to this study. The NIH Institute funded this project through grant, R01CA190182, awarded to G.A. Coetzee. Van Andel Institute also provided the facilities and general support to conduct our research. Marie Adams and the rest of the Genomics Core conducted and advised on next generation sequencing and Lee Marshall provided analysis advice.

Research reported in this publication was supported by the National Cancer Institute (NCI) of the National Institutes of Health under award number, R01CA190182. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

COI: No potential conflict of interest was reported by the authors.

Ethics approval

Human cell lines used in this study were obtained from ATCC and do not require additional ethical approval. All other data used was obtained from public sources.

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Effect of short-term fasting on the cisplatin activity in human oral squamous cell carcinoma cell line HN5 and chemotherapy side effects

Nafiseh Sheykhbahaei 1 ,
Ahmed Hayder AL Tameemi 2 &
Maryam Koopaie ORCID: orcid.org/0000-0002-9999-1443 1

BMC Cancer volume 24 , Article number: 989 ( 2024 ) Cite this article

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Ketogenic interventions like short-term fasting show potential as complementary therapies to enhance the effectiveness of chemotherapy for cancer. However, the specific effects of fasting on head and neck squamous cell carcinoma (HNSCC) cells and healthy oral mucosa cells during these treatments are not well understood. This study investigates whether short-term fasting can differentially impact HNSCC cell survival and viability compared to healthy keratinocytes while undergoing standard chemotherapy regimens.

This study investigated the effects of fasting on cell viability in HN5 cell line and healthy oral keratinocyte cells. The HN5 cell line, derived from human tongue squamous cell carcinoma, and primary human keratinocytes isolated from the basal layer of gingival epithelium were divided into three groups: (1) control, (2) treated with the standard chemotherapeutic agent cisplatin, and (3) treated with cisplatin under fasting conditions achieved through 48-hour glucose restriction mimicking the blood glucose levels of fasted individuals. Cell proliferation was assessed at 48 and 72 h using the MTT assay, a colorimetric method based on mitochondrial dehydrogenase activity. Flow cytometry analysis with specific apoptosis and necrosis markers distinguished between early and late apoptotic, necrotic, and viable cells.

Cell viability in HN5 and healthy keratinocyte cells decreased in cisplatin with low glucose groups compared to cisplatin and control groups. The same results were observed for healthy keratinocyte cells; only a decrease in cell viability in cisplatin groups compared to control groups was observed, which was not statistically significant. Cell apoptosis in HN5 and healthy keratinocyte cells increased in cisplatin with low glucose groups compared to cisplatin and control groups. In healthy keratinocyte cells, the cisplatin with low glucose group showed an impressive increase in necrosis, late apoptosis, and early apoptosis and a significant decrease in live cells compared with other groups.

This study revealed that short-term fasting chemotherapy significantly improved HNSCC cell line apoptosis and necrosis.

Peer Review reports

Introduction

The prevalence of cancer is increasing worldwide, bringing considerable costs to healthcare systems. Cancer is the second leading cause of death worldwide [ 1 ]. Head and neck SCC is the seventh most common cancer diagnosis worldwide [ 2 ]. Tobacco use, excessive alcohol consumption, human papillomavirus (HPV) infection, betel quid chewing, and poor diet are well-established risk factors for oral cancer development [ 3 , 4 , 5 ]. Notably, modern diets, often high in refined carbohydrates, have been implicated in carcinogenesis [ 6 ]. Conversely, the ketogenic diet, which restricts carbohydrates and promotes fat metabolism, may create an unfavorable microenvironment for cancer cells [ 7 ].

Fasting, particularly short-term fasting (STF), has emerged as a promising strategy to enhance cancer treatment outcomes [ 8 ]. Studies suggest fasting can augment cancer cell sensitivity to chemotherapy while protecting healthy cells [ 9 , 10 ]. This effect is likely attributed to a metabolic shift in cancer cells towards less efficient energy production pathways than healthy cells [ 11 ]. The use of conventional treatments, including surgery, radiotherapy, and chemotherapy drugs, remains the most accepted approach among physicians and patients. This is due to their easy access, lower cost, and effectiveness against various cancers. While newer approaches like targeted therapy drugs hold promise and demonstrate effectiveness in cancer treatment [ 12 , 13 ], their application is limited to specific cancer subgroups. Additionally, these therapies come with a high cost and pose access challenges [ 14 , 15 ]. Chemotherapy and radiotherapy destroy cancer cells through genotoxicity, which is the production of reactive oxygen species (ROS) [ 16 ]. Healthy cells are also likely to be destroyed and severely damaged, causing side effects such as bone marrow suppression, fatigue, nausea, diarrhea, oral mucositis, and even death. Despite the various treatment modalities used for HNSCC, the survival rate for the disease remains relatively low, with a 5-year survival rate of 50–60% [ 17 , 18 ].

Due to the limited effectiveness of common treatments caused by toxicity in healthy tissues, recent research has focused on developing adjuvant strategies utilizing chemoproteomic agents [ 19 , 20 , 21 ]. These agents exploit differential effects to selectively increase tumor cell sensitivity to treatment while enhancing the resistance of healthy cells to toxicity [ 22 ]. Most cancers, especially carcinomas of epithelial origin, appear to be caused by a disturbance in metabolism associated with the modern lifestyle [ 23 ]. Diets high in carbohydrates, a common feature of modern lifestyles, can induce epigenetic modifications through specific chromatin conformations and alterations in DNA structure. These epigenetic changes can impact genomic stability and the production of proteins and mRNA, potentially contributing to metabolic disorders [ 24 ]. Modern lifestyles have a significant impact on the development of breast, cervical, oral, and gastric cancers, among others [ 25 ]. Another aspect of cancer treatment is the use of the ketogenic diet as an adjuvant therapy. This diet likely creates an unfavorable metabolic environment for cancer cells, making it a promising candidate for patient-specific, multifactorial treatment approaches. Some cancer cells, due to mitochondrial dysfunction, lack the ability to metabolize ketone bodies produced by the ketogenic diet. Additionally, the diet reduces blood glucose levels, which can lead to a decrease in insulin and insulin-like growth factor (IGF), both of which are important drivers of cancer cell proliferation [ 26 , 27 ]. A rapid increase in carbohydrate consumption plays a significant role in these metabolic changes and tumorigenesis [ 28 ]. Tumor tissue consumes significantly more glucose than healthy tissue. Even in the presence of oxygen (aerobic conditions), tumor cells produce large amounts of lactate. This is a key difference from healthy tissue, which minimizes lactate production through respiration [ 29 ]. Therefore, limiting or eliminating glucose sources through a ketogenic diet has been explored as a potential approach for cancer patients [ 30 , 31 ]. Previous research supports the idea that ketogenic interventions, such as fasting, could be effective adjuncts to improve the outcomes of radiotherapy and chemotherapy [ 32 , 33 , 34 , 35 ]. Fasting enhances the sensitivity of cancer cells to chemotherapy during fasting [ 23 ]. This effect is likely due to a combined increase in ROS production and a decrease in adenosine triphosphate (ATP) levels, caused by a metabolic shift from glycolysis to mitochondrial metabolism in tumor cells [ 36 ]. During fasting, healthy cells undergo a metabolic switch from growth to maintenance mode. This involves a decrease in glucose and growth factor availability, accompanied by an increase in ketone bodies. Ketone bodies serve as an alternative fuel source and are associated with reduced cellular damage in healthy tissues [ 37 ]. The metabolic difference underlies the differential stress resistance phenomenon (DSRP) in healthy and cancer cells [ 38 ]. STF is a dietary approach that involves completely restricting calorie intake for a limited period of time. STF is considered a non-invasive and cost-effective complementary treatment option [ 39 ]. Several studies have shown that STF increases a specific type of cancer cell death, while also protecting healthy cells during chemotherapy. This effect has been observed in both laboratory in-vitro and in-vivo studies [ 40 , 41 , 42 , 43 , 44 , 45 , 46 ]. However, no study has evaluated the effects of fasting on head and neck squamous cell carcinoma (HNSCC) and healthy oral mucosa cells under chemotherapy. The available preclinical and limited clinical data suggest that intermittent fasting may have favorable effects on breast cancer outcomes [ 47 ]. Diabetic-FBG (fasting blood glucose) level was found to be an independent prognostic factor for patients with oral cancer [ 48 , 49 ]. Fasting or fasting-mimicking diets have shown potential in modulating the tumor biology and improving the effects of cancer therapies in brain tumors [ 50 ] but more robust clinical studies are needed to confirm these findings.

SCC is the most common malignant tumor of the head and neck [ 51 ]. It is associated with a low life expectancy and often shows a poor response to treatment, exhibiting high resistance. Surgery, radiotherapy, and chemotherapy (for distant metastasis) are the main treatment options for HNSCC [ 51 ]. In many cases, significant side effects of chemotherapy, such as oral mucositis, can disrupt the treatment process [ 52 , 53 , 54 , 55 ]. Therefore, it is necessary to identify methods that increase cancer cells’ destruction while reducing conventional therapies’ toxicity on healthy cells. Therefore, this study was designed to investigate the effect of STF on HNSCC cells and healthy keratinocyte cells under chemotherapy.

Ethical statement

The protocol of this experimental and interventional in-vitro study was approved by the Ethics Committee of Tehran University of Medical Sciences (ethical code: IR.TUMS.DENTISTRY.REC.1400.124) and Informed consent was obtained from all individuals. All methods were performed in accordance with the relevant guidelines and regulations and this study was conducted in accordance with the Declaration of Helsinki [ 56 ].

Cell culture

The HN5 cell line (code: NCBI, 30196) was purchased from the National Cell Bank of Iran, affiliated with the Pasteur Institute of Tehran, Iran. This cancer cell line was derived from a tongue squamous cell carcinoma in a 73-year-old man (Supplementary file 1 ). The cells were cultured in a Dulbecco’s Modified Eagle Medium (DMEM) (BIOSERA, USA) that contains 10% Fetal Bovine Serum (FBS) (GIBCO, USA). They were cultured in a cellular incubator for proliferation to reach a suitable density at 37 °C and 5% CO 2 and 1-2% humidity for 24 h. Pen/strep (ATOCEL Company, Budapest), a solution containing standard antibiotics, penicillin, and streptomycin, was added to prevent the growth of a variety of Gram-positive and Gram‐negative bacteria.

Human healthy keratinocyte cell was obtained from gingival tissue removed under sterile conditions for crown lengthening, then quickly transferred to the laboratory in a phosphate-buffered saline (PBS) container along with 3% pen/strep antibiotic in the vicinity of a 4 °C ice pack. The sample was washed several times with hank buffers containing antibiotics in the laboratory and under sterile conditions below the hood, then the target tissue was placed in tissue solvent solution including DMEM (BIOSERA, USA) and collagenase type 1 (Worthington Biochem, Freehold, NJ, USA) with concentration 250 U/ml and was incubated in a shaking incubator for 1 to 2 h at 37 °C and 5% CO 2 for tissue digestion. After the tissue was digested, filtration was performed with 70 μm filters (Falcon, BDL labware, Franklin Lakes, NJ, USA) to remove undigested tissue fragments and impurities. Afterward, the solution containing the sample was centrifuged in at 1500 rpm for 5 minutes to form a cell plate. Then, the cells were cultured in a T75 flask (Greiner, Frickenhausen, Germany) containing DMEM 80%/ F12 medium (BioChrom AG, Berlin, Germany) with 10% FBS (GIBCO, USA) and 10% pen/strep. The culture medium has been changed every two days. And each week it’s got it-term trypsinization for 5 minutes by 1 mL of Trypsin-EDTA (Sigma-German) solution (Trypsin 0.25% and one molar EDTA), the cells got separated and cultured in a new flask with a density of 1 × 10 5 cells/cm².

After Trypsinization of the cells and centrifuging and suspending the cell’s sediment in one milliliter of culture medium, cell counting was done. Then, according to the number of cells obtained in a volume of 1000 µl, using a simple ratio, a volume of suspension containing 5000 cells was calculated. According to the calculations, 5000 cells were cultured in each well of a 96-well plate (in 3 groups of 3 replicates) and the cells were placed in an incubator for 24 h.

SCC cells are divided into three groups; Cells without treatment (SCC-1), under treatment with a standard chemotherapeutic agent (SCC-2), and cells under treatment with standard a chemotherapeutic agent and fasting condition (SCC-3). Healthy oral keratinocyte cells are divided into three groups; Cells without treatment (Healthy-1), under treatment with a standard chemotherapeutic agent (Healthy-2), and cells under treatment with standard chemotherapeutic agent and fasting condition (Healthy-3).

In vitro fasting (short-term fasting)

Cellular fasting was done by glucose restriction to achieve blood glucose levels typical of fasted and healthy cells; for human cell lines, cells were washed twice with PBS before changing to a fasting medium. For Short-Term fasting medium (STF), cells were grown in DMEM medium without glucose (DMEM no glucose, Life Technologies, Cat. No. 11966025) supplemented with 0.5 g/L glucose (Sigma-Aldrich, Cat. No. G8769) and 1% FBS. For mimicking normal conditions, cells were grown in a DMEM medium without glucose (DMEM no glucose, Life Technologies, Cat. No. 11966025) supplemented with 1 g/L glucose (Sigma-Aldrich, Cat. No. G8769) and 10% FBS, referred to as control medium (CTR) [ 57 ]. All treatments were perform at 37 °C under 5% CO 2 . Forty-eight hours later, a chemotherapeutic agent was added to culture media.

Chemotherapeutic agent preparation

10 mg/mL vials of 1 mg/mL cisplatin were purchased from Mylan Company, France. In-vitro chemotherapy was performed by treating cells in a medium containing cisplatin with a dose of 8 µl/ml for 48 h.

Cell proliferation assay

After 48 and 72 h, cell proliferation was measured using the 3-(4,5‐dimethylthiazol‐2‐yl) ‐2,5‐diphenyltetrazolium bromide (MTT) test. MTT powder was combined with PBS medium, and the resultant solution was applied to the wells of a 96-well microplate that contained cell lines and groups. After a four-hour incubation at 37 degrees Celsius, dimethyl sulfoxide was supplied to the wells, and the absorption rate was recorded at 570 nanometers.

Annexin V-FITC/PI staining and flow cytometry

After treatment with fasting and cisplatin alone or in combination, the cells were trypsinized and incubated with Annexin V–conjugated MicroBeads (Miltenyi Biotec GmbH, Germany), according to the manufacturers protocol. The apoptosis rate was then assessed using a FACS Calibur flow cytometer (BioTed USA), and the data was analyzed using Cell Quest (BD Biosciences) and FlowJo (Tree Star Inc., Ashland, OR, USA) software. According to the staining profile, the early apoptotic cells (FITC+ /PI), late apoptotic cells (FITC /PI+), necrotic cells (FITC+ /PI+), and intact cells (FITC /PI) were discriminated.

Statistical analysis

The data were analyzed using SPSS (version 22.0; SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8.2.1 (GraphPad Software, San Diego, CA). One-way ANOVA was used to compare the means of different groups, followed by Tukey HSD post hoc analysis for pairwise comparisons. A p-value (p) less than 0.05 was considered statistically significant.

Cell viability in HN5 and healthy keratinocyte cells

Based on the 48 and 72-hour MTT analysis, the control groups (SCC-1 and Healthy-1) exhibited the highest cell viability (100%) for both HN5 and healthy keratinocyte cells. Cell viability was followed by the cisplatin-treated groups (SCC-2 and Healthy-2). The lowest average cell viability was observed in the groups treated with cisplatin under low glucose conditions (SCC-3 and Healthy-3), (Supplementary file 2 and 3 ) (Tables 1 and 2 ).

One-way ANOVA analysis revealed a statistically significant difference ( p < 0.05) in cell viability between groups for both HN5 and healthy keratinocyte cells at both time points. Specifically, in HN5 cells, significant differences were observed at 48 h ( p < 0.001, F = 87.764) and 72 h ( p < 0.001, F = 88.793) of MTT assay. Similarly, significant differences were found in healthy keratinocytes at 48 h ( p = 0.002, F = 20.629) and 72 h ( p < 0.001, F = 53.213) of MTT assay.

Tukey’s HSD analysis in HN5 cells showed a statistically significant decrease in cell viability in cisplatin groups (SCC-2) ( p < 0.05) and cisplatin with low glucose groups (SCC-3) ( p < 0.05) to control groups (SCC-1). Also, the decrease in cell viability in the cisplatin with low glucose groups (SCC-3) to cisplatin groups (SCC-2) was statistically significant (Table 1 ; Fig. 1 -A) ( p < 0.05). These results were found in both 48 and 72 h of MTT assay. These same results were also reported for the healthy keratinocyte cells; only a decrease in cell viability in cisplatin groups (Healthy-2) to control groups Healthy-1) was observed which not statistically significant ( p = 0.421) (Table 2 ; Fig. 1 -B).

MTT assay of cell viability: A ) HN5 cell line after 48 and 72 h, B ) healthy keratinocyte cells after 48 and 72 h. ns: p value > 0.05, *: p value ≤ 0.05, **: p value ≤ 0.01, ***: p value ≤ 0.001

Cell apoptosis in HN5 and healthy keratinocyte cells

According to the Annexin-V test in the HN5 cells, the highest cell necrosis average was in the cisplatin group (SCC-2), the highest average of late apoptosis and early apoptosis, and the lowest average of live cells was related to cisplatin with low glucose groups (SCC-3). Relatively similar results were obtained in healthy keratinocyte cells. The highest mean of cell necrosis, late apoptosis, and early apoptosis and the lowest average of live cells were observed in the cisplatin with low glucose groups (Healthy-3) (Tables 3 and 4 ). The results of the one-way ANOVA analysis showed a statistically significant difference in the percentage of cell necrosis ( p = 0.024, F = 7.452), late apoptosis ( p < 0.001, F = 82.506), early apoptosis ( p = 0.001, F = 34.231) and live ( p < 0.001, F = 254.450) cells between the groups of HN5 cells, and significant difference in the percentage of cell necrosis ( p = 0.182, F = 2.297), late apoptosis ( p = 0.033, F = 6.307), early apoptosis ( p = 0.274, F = 1.619) and live ( p = 0.009, F = 11.225) cells between the groups of healthy keratinocytes ( p < 0.05). These results were found in both 48 and 72 h MTT assay (Table 3 ; Fig. 2 ).

Apoptosis results of HN5 cell line in in various group of treatment

Tukey’s HSD analysis of HN5 cells revealed a statistically significant increase in both late and early apoptosis ( p < 0.05) in the cisplatin with low glucose group (SCC-3) compared to other groups (Tables 3 and 4 ; Fig. 2 ). Additionally, a significant decrease in live cells was observed in the cisplatin with low glucose group (SCC-3) ( p < 0.05). In contrast, cell necrosis was statistically higher only in the cisplatin group (SCC-2) compared to the control group, with no significant difference observed in the cisplatin with low glucose group (SCC-3). These findings were consistent at both 48 and 72 h as measured by the MTT assay.

For healthy keratinocyte cells, the cisplatin with low glucose group (Healthy-3) displayed a significant increase in necrosis, late apoptosis, and early apoptosis compared to other groups ( p < 0.05) (Table 4 ; Fig. 3 ). This was accompanied by a significant decrease in the number of live cells.

Apoptosis results of healthy Keratinocyte cells in various group of treatment

For the first time, this study investigated the effect of STF on the HNSCC cell line. Based on the results of the study, short-term fasting for 48 h before chemotherapy significantly decreases the cell viability and increases the apoptosis of HNSCC cells compared to the control group. However, the protective effect of this adjuvant method was not seen in preserving healthy keratinocyte cells against chemotherapy. Warburg’s effect refers to the chemoresistance of the tumor, associated with high aerobic glycolysis and low oxidative phosphorylation [ 58 ]. Tumor-starved cells promote an anti-Warburg effect through increase in the translation of selected genes such as the AKT/S6K signaling pathway, which leads to an increase in cell respiration and oxygen consumption. This causes increased ROS, DNA damage, Caspase 3 activation, and apoptosis, especially during chemotherapy [ 10 , 16 , 59 ].

Therefore, declines in plasma levels of insulin-like growth factor-1 (IGF-1), insulin, and glucose mediate the effects of fasting on cancer cells by improvement of apoptosis. Since the reduction of glucose and IGF1 are the main mediators in the effectiveness of ketogenic therapy on cancer and healthy cells, the STF diet seems to be preferable to caloric restriction (CR) or intermittent fasting (IF) [ 9 , 28 ]. Two studies showed that IF with or without CR did not significantly improve survival and reduce tumor growth in mice with prostate cancer [ 36 , 37 ]. It is noteworthy that the decrease in blood glucose level after STF (2–5 days) is 75%, and the decrease in IGF-1 was 75%; however, after long CR or IF, only a 15% decrease in blood glucose and 25–30% decrease in IGF-1 have been reported [ 9 ].

Several previous studies confirmed the beneficial effects of short-term fasting during chemotherapy and radiotherapy [ 10 , 16 , 28 ]. Fasting adjuvant to chemotherapy drugs causes a significant reduction in tumor size, tumor weight, tumor bioluminescence, adenosine levels in the micro tumor environment, and upregulation of autophagy, which can prevent tumor progression and improve anti-tumor immunity in various cancer cell lines and higher survival rate in animal mode. Consistent with our findings, Lee et al. indicated higher apoptosis in glioma cells and 4T1 breast cancer cells following 48 and 72 h of STF before chemotherapy, respectively. However, in contrast to our result, he showed decreased apoptosis in healthy glia cells [ 10 , 28 ]. Bianchi et al. also established that 48 h of STF can promote apoptosis in colon tumor cell lines [ 16 ].

In addition to laboratory and animal studies, some human studies have also shown the positive effects of fasting therapy in cancer treatment. De Groot et al. showed that a fasting-mimicking diet for four days before chemotherapy led to better therapeutic effects and radiological evidence than the control group [ 16 ]. According to the DSRP theory, fasting can improve the survival and preservation of healthy cells against the toxic effects of chemotherapy drugs. This effect was not confirmed on healthy keratinocyte cells in the current study. De Groot et al., in a pre-clinical study, stated that STF (24 h before and 24 h after) significantly protected from the hematological toxicity associated with chemotherapy. However, non-hematological toxicity, including fatigue, infection, mucositis, neuropathy, diarrhea, dizziness, nausea, constipation, and eye problems, did not differ between the two groups. They suggested that the reduction of hematotoxicity can be related to the lower intensity of bone marrow suppression or the reduction of the breakdown of circulating blood cells and possibly the faster recovery of DNA damage in peripheral blood mononuclear cells after chemotherapy in the Fasting group [ 60 ]. In other human studies, with increasing STF duration, especially before chemotherapy (48, 36, and 72 h), we see a reduction in hematological and non-hematological toxicity and an improvement in quality of life in treated patients [ 61 , 62 , 63 ]. Reducing the non-hematologic toxicity of chemotherapy compared to hematologic toxicity requires a relatively long fasting period, especially before the chemotherapy, and subsequently, a more significant reduction in the level of IGF-1. The reduction of chemotherapy side effects by STF has been reported even in studies that did not significantly improve the antitumor effects of chemotherapy. The strong reduction of active metabolites of chemotherapy drugs in the serum and liver of fasting group mice, despite the lack of difference in tumoral tissue [ 64 ], can justify the lower side effects and, simultaneously, similar therapeutic effects in the Fasting group.

Building on the findings of this study and aligning with the majority of previous research, STF appears to significantly enhance cancer treatment. Even in studies without statistically significant differences in treatment outcomes, STF did not impede chemotherapy’s ability to reduce tumor size or markers. Furthermore, the potential reduction of chemotherapy side effects strengthens the case for STF as an adjuvant therapy. Importantly, all human and animal studies report good tolerability and safety with the STF diet. Therefore, we propose further large-scale clinical trials investigating the effects of different STF durations and adjuvant chemotherapy drugs on tumor toxicity, along with hematological and non-hematological side effects. This research should encompass a diverse patient population representing various stages of HNSCC.

The finding of this study revealed that short-term fasting (48 h before and 48 h after) chemotherapy significantly improved HNSCC cell line apoptosis and necrosis; however, the protective effect of fasting therapy on healthy oral keratinocytes has not been established. This study lays the groundwork for future research avenues exploring the potential of Short-Term Fasting (STF) as a therapeutic strategy for HNSCC. Future investigations should delve deeper into the mechanism of action by employing Western blot analysis to pinpoint changes in apoptosis and cell cycle proteins. Additionally, studies with extended durations and varying fasting periods are warranted to assess long-term effects and dose-response relationships. Furthermore, exploring the potential of combining STF with chemotherapy drugs for synergistic anti-cancer effects or reduced toxicity on healthy cells holds promise. Finally, investigating the impact of STF on cell migration and invasion, as well as cytokine and inflammatory marker profiles, could provide valuable insights into its influence on the tumor microenvironment and its potential immunomodulatory properties. These future directions offer exciting possibilities for harnessing the power of STF in the fight against HNSCC.

Data availability

The data supporting the findings of this study are available upon reasonable request from the authors.

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Acknowledgements

This study has been supported and funded by the Tehran University of Medical Sciences (TUMS). Grant number: 1400-3-219-55145.

This study has been supported and funded by Tehran University of Medical Science (TUMS). Grant number: 1400-3-219-55145.

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Nafiseh Sheykhbahaei & Maryam Koopaie

Dentist, Department of Oral Medicine, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran

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N. Sh: Substantial contribution to the conception and design, drafting of the manuscript, critically revising the manuscript for important intellectual content, approval of the final version submitted for publication. “Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.”M. K: Substantial contribution to the analysis and interpretation of data, drafting the manuscript, and approval of the final version submitted for publication. “Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.”A H. A T: Substantial contribution to the acquisition and analysis of data, critically revising the manuscript for important intellectual content, and approval of the final version submitted for publication. “Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.”

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Correspondence to Maryam Koopaie .

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Sheykhbahaei, N., Tameemi, A.H.A. & Koopaie, M. Effect of short-term fasting on the cisplatin activity in human oral squamous cell carcinoma cell line HN5 and chemotherapy side effects. BMC Cancer 24 , 989 (2024). https://doi.org/10.1186/s12885-024-12752-2

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A positive increase in micronuclei in mammalian binucleate cells was registered for MCL-5 cells exposed to both Slick (0.5 mg/ml) and Crude oil (0.063 mg/ml) and a positive increase in micronucleated binucleate cells was also presented in both cell lines when exposed to either DPM or TMP.
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CELL LINE: A REVIEW

Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies

Conclusions

Cell lines – Culturing, processing and analyses overview

Targeted deep sequencing

High resolution DNA copy number profiles

Gene expression analysis

Small RNA sequencing

High-throughput protein expression analysis

Integration of DNA copy number and gene expression data

DNA sequence aberrations reflect hypermutator phenotypes

DNA copy number aberrations reflect the CIN phenotype

mRNA, miRNA and protein expression profiles are distinct between undifferentiated and “colon-like” cell lines

Integrated analysis identifies in vitro models for studies of targetable genes

Concurrent CNAs and SNVs/indels in cancer critical genes

mRNA expression of oncogenes and transcription factors is associated with DNA copy number in cis

Gene amplification and outlier expression

Consistent expression regulation at the gene and protein level

Multi-level data integration emphasizes molecular differences among cell lines relevant for functional studies

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Box 1 Definitions of terms frequently used in tissue culture

1. Cell line development, acquisition and authentication

1.1. Developing a new cell line

1.1.1. Tissue

1.1.2. Clinical information

1.1.3. Accessory information

1.1.4. Cell line designation

1.1.5. Publication

1.2. Acquiring a cell line from another laboratory

1.2.1. Quarantine

1.2.2. Authentication

1.2.3. Characterisation

1.3. Cell banks

1.4. Storage and banking

1.4.1. Cryopreservation

1.4.2. Storage

1.5. Cell line misidentification

1.5.1. Recognising cell line misidentification

1.6. Cell line distribution

1.6.2. Transport containers

1.6.3. Practicalities

1.7. Regulations for the transport of cells

1.7.1. Within the UK

1.7.2. Import to the UK