Misidentified and contaminated cell lines are not a new problem. Far from it. Alarms about the shortcomings of many cell lines and their deleterious impact on experiment reproducibility and the scientific record have been ringing for over 50 years. One of the earliest warnings was from Stanley Gartler, Ph.D., a professor at the University of Washington, who reported at a National Cancer Institute (NCI) meeting in 1966 that many cell lines he examined were contaminated with HeLa cells.

Gartler, a geneticist by training, was denounced for his lack of cell culture expertise and, in what must have been a profane 60s putdown, was said to have “dropped a turd in the punch bowl” at the meeting. Predictably, his discovery was not accepted or appreciated by the cell culture community, and for many decades his evidence was ignored.

Today, even though there is consensus among scientists that misidentified and contaminated cell lines are a significant problem, some labs are reluctant to authenticate their cell lines.

Christopher Korch, Ph.D., from the University of Colorado, believes this is a multifaceted problem. “I ascribe this attitude to (1) a lack of proper cell culture training by the PI [principal investigator], (2) not understanding the enormity of the problem, (3) not realizing how easily contamination can occur, and (4) the assumption that one does not make mistakes.”

But mistakes are being made and they are piling up. Korch reported in 2015 that nearly 5,800 articles in 1,182 journals may have confused HeLa for HEp-2, and another 1,336 articles in 271 journals may have mixed up HeLa with INT 407. "We're looking at tens of thousands of publications, millions of journal citations, and potentially hundreds of millions of research dollars," he told Science.

Korch adds that he regularly tries to “instill in my colleagues an understanding of how easy it is for cell lines to cross contaminate each other.” He mentions research by Lewis Coriell, M.D., Ph.D., that “showed that HeLa cells could be easily dispersed in aerosols and spread by other means, namely they could ‘fly’.” In addition, Korch recommends that scientists read an article by Geraghty et al., published in 2014 in the British Journal of Cancer. “There are many useful suggestions of how to avoid the problem that are discussed in the extensive article.”

Comprehensive training is important

Comprehensive training on issues surrounding scientific reproducibility, including cell-line authentication and antibody validation, will certainly help resolve the problem, but there are other elements of the life science research machine that are ripe for improvement as well. Korch, an active member of The International Cell Line Authentication Committee (ICLAC), cites calls to action for the four major stakeholders.

  • Researchers and their laboratories: “They need to change their attitude about the importance of regular cell-line authentication and understand its utility and limitations. This includes doing it before starting a project, during a project, and before submitting a manuscript or grant.”
  • Research institutions: “They need to establish cell-line authentication policies so that they avoid having research articles retracted because of cell-line issues. Retractions can result in loss of research funding, legal issues, and more.”
  • Journals: “Their submission policies must be strengthened in regards to cell-line policies. The best policy is that of the International Journal of Cancer. These policies should not include the option to simply declare that the cell lines were not authenticated as is done by the AACR [American Association for Cancer Research] journals. They must implement a checklist and must train their reviewers and editors to critically evaluate manuscripts for cell-line authenticity using among others the ICLAC list of imposter cell lines, Cellosaurus, and Research Resource identifiers (RRIDs) for their reagents.”
  • Granting Agencies: “The 2016 NIH [National Institutes of Health] policy targeting reproducibility is one that can be implemented by other funding agencies. The grant reviewing procedure must include a checklist and training of their reviewers to critically evaluate manuscripts for cell line authenticity using among others the ICLAC list of imposter cell lines, Cellosaurus, and use RRID [Research Resource Identifiers] identifiers for their reagents.”

Testing methods help identify cell line integrity

Short tandem repeat (STR) genotyping is the method of choice to authenticate cell lines for several reasons. This technology was well developed by the forensic field, and the kits that are commercially available are well validated. In addition, databases have been set up with STR profiles based on these kits so that the results from different laboratories can be compared to reference profiles. “The DSMZ [Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures] and the ATCC [American Type Culture Collection] have freely accessible large collections of STR profiles and online search engines to find profile matches,” Korch notes.

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Another technique to ensure the quality and integrity of human cell lines is single-nucleotide polymorphism (SNP) assays, which can complement STR genotyping results. SNP genotyping assays have both advantages and disadvantages in comparison to STR-based assays. “Some cancer cell lines that come from endometrial or intestinal cancers can have defects in the cell's DNA mismatch repair system and as a consequence gives rise to instability of microsatellite sequences, which in turn can result in variations in the STR profiles,” Korch says.

SNPs are useful when comparing a cell line that was examined for mutations in a gene, but which was not STR genotyped. Thus, sequencing specific SNPs can confirm whether the cell line has the same mutation that was reported earlier for an early sample of the cell line or for the tumor from which the cell line was supposedly derived.

The major problem with SNP genotyping is that currently there does not exist a searchable database for identifying cell lines like that available for STR genotyping of cell lines.

According to Korch, next-generation sequencing assays of both STRs and SNPs in samples are being developed. Once validated by the forensic community, they will have the advantages of both SNP and STR assays, “Plus they will allow comparison to the older STR data, which SNP data alone cannot provide.”

The role of ICLAC

ICLAC was formed in 2012 to make cell-line misidentification more visible and to promote awareness and authentication testing as effective ways to combat it. Among the resources on the organization’s site are several “Online Databases of Human Cell Line STR Profiles.” Amanda Capes-Davis, Ph.D., who has written extensively on the topic, is ICLAC chair. Korch is a member, as is Roland Nardone, Ph.D., a long-time cell-line authentication provocateur. ICLAC’s website lists about 500 cell lines that are known to be misidentified with links to the Cellosaurus website, which lists 100,000 cell lines.

Several members of ICLAC have also been involved in the development of the ATCC ANSI Standards describing (1) how to authenticate human cell lines and (2) how to identify animal species by the Barcode of Life based on the mitochondrial cytochrome c oxidase subunit 1 (COX 1) gene. In addition, members have had a hand in some 60 articles about cell-line contamination over the last six years.

Capes-Davis, Korch, and other ICLAC members are not resting on their laurels, however, or basking in the increased awareness of the importance of cell-line authentication. They believe there is much more to be done, including more extensive training programs and convincing institutions to adopt stringent cell-line authentication policies similar to those from MD Anderson Cancer Center.

Moving forward

Many members of ICLAC as well as Stanley Gartler, Len Freedman, Ph.D., from Global Biological Standards Institute (GBSI), and representatives from biopharma, life science tool companies, and research institutes are featured in Biocompare’s recently launched Cell Line Authentication documentary.  This documentary not only reviews the problem of cell-line authentication, it also discusses solutions and recommendations for the future, including best practices to combat misidentified and contaminated cell lines as well as ways researchers can pay it forward when developing new cell lines.