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Ending cell line contamination by cutting off researchers | Top Cell Culture Feature of 2010

08/10/2010
Erin Podolak

After 50 years of skepticism, finger pointing, and unenforced protocols, sentiments are growing for mandatory cell line authentication as a condition for funding and publication. Erin Podolak investigates the current state of cell line contamination and finds how raising awareness could help cut off the supply of contaminated lines.

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When the first human cancer cell line was established from Henrietta Lacks in 1952 (1), the biological community didn’t foresee the enduring impact that the highly celebrated HeLa cell line would have. But these fast-growing cervical cancer cells are perhaps the most well-known example of cell line contamination (2). The contamination problem, which the biological research community has been aware of for decades, goes far beyond HeLa cells. Still, no major group or movement has succeeded in stopping the distribution of contaminated cell lines, and the publication of tainted research from those lines continues to be unmitigated.

HeLa cells, imaged using phase contract micrscopy, are perhaps the most well-known example of cell line contamination. Source: MicroscopyU.com Phase Contrast and DIC Comparison Image Gallery

“This has not been a high profile issue for either the public or the research community,” says Amanda Capes-Davis, a member of the American Type Culture Collection (ATCC) Standards Development Organization (SDO) working group, which is developing standards for human cell line authentication. “Hopefully though, for the research community that is gradually changing.”

To date, there have been no mandatory rules established by major research funding agencies—namely the National Institutes of Health (NIH)—or any of the most popular research journals regarding cell line authentication. But with the ATCC-SDO expected to issue a report on standards for human cell line authentication later this year, some scientists think the research community’s reticence to adopt strict authentication protocols may be coming to a forced end.

Where did it all go wrong?
“After HeLa, it seemed much easier to establish cell lines and everyone started to do it,” says Capes-Davis, who is also the founding manager of Cell Bank Australia. “Then in the late 1960s and early 1970s, Stanley Gartler and Walter Nelson-Rees showed that many later cell lines had been overgrown by HeLa, most likely during establishment.”

Even though the work of Gartler and Nelson-Rees (3-4) exposed HeLa cell contamination nearly 40 years ago, the use of HeLa-contaminated lines is a persistent problem. “Many are still used by laboratories all over the world as if they were authentic, because the original publication exposing the contamination was many years ago,” explains Capes-Davis. “It often goes unnoticed.”

It is well documented that HeLa cells are responsible for a significant amount of contamination, namely in the intestinal epithelium (Int-407), normal amnion (WISH), normal liver (Chang liver), laryngeal cancer (Hep-2), and oral cancer (KB) lines. But the problem stretches beyond HeLa. For example, the normal endothelial cell line ECV304 has been shown to be contaminated with T24 bladder cancer cells, but it’s still accepted as a model for endothelial cells (5). The human prostate cancer cell lines TSU-Pr1 and JCA-1 are also actually T24 bladder cancer cells (6), and the breast cancer line NCI-ADR-RES is in fact the ovarian tumor line OVCAR-8; errant data on this line has been published over 300 times (7).

All in: research is a high-stakes game
In 2009, researchers from the University of California, San Francisco (UCSF) published a study showing that the adenoid cystic carcinoma cell lines ACC2, ACC3, ACCM, ACCNS, ACCS, and CAC2 have all been subject to contamination and misidentification (8). Led by Osamu Tetsu, a salivary gland researcher at UCSF, the study found that these contaminated cell lines were widely used. But according to Tetsu, publishing this fact has done little to curb their use.

“After publication of this paper, my colleagues recognized the problem, but they are [still] reluctant to check authentication of their own cell lines,” says Tetsu. “They overwhelmingly say they cannot do so because they are afraid of losing their data, and the chance of publication as a result of cell contamination.”

Though the risk of losing data may seem like an unreasonable explanation for why researchers continue to work with contaminated cell lines, Tetsu says the setback to a researcher’s career could be devastating. “Say the work in question is done by a graduate school student, and he or she finds out about the problem in their last year of the study. They lose not only the data but also that precious time in their professional life.”.

Richard Badge, from the department of genomics at the University of Leicester in the UK, works on developing ways to identify cell line contamination (9). Badge says that the very nature of science as a business contributes to perpetuating the use of contaminated cell lines. “I think the main issue is the ‘publish or perish’ climate endemic in science today. For researchers who have established a body of work based on a particular cell type, for that cell line to be revealed as non-authentic would be disastrous. I think this has led to a head-in-the-sand mentality.”

Stopping researchers before they start
So what is a researcher to do? Torpedo their own career by admitting to working with contaminated cell lines, thus losing years of research, their reputation, and future funding prospects? Unlikely, says Capes-Davis. But stopping researchers before they start could help eliminate research based on known contaminated cell lines. According to Capes-Davis, there is a push in the research community to continue spreading the word, so researchers will know not to start working with these cell lines in the first place.

“Individuals within the research community have acknowledged the problem, and many have put in enormous efforts to improve the situation. Cell line contamination is not an area that normally gets funded, so in most cases their contribution has been driven by a pure desire to improve the situation,” says Capes-Davis.

To this end, Capes-Davis works closely with colleague Ian Freshney, of the Centre for Oncology and Applied Pharmacology at Glasgow University, to maintain a list of contaminated cell lines (10). “This was one of the main reasons my colleagues and I wanted to develop a list of these cell lines,” she explains, “to make it easier for laboratories to check for references before they put effort into working with a contaminated cell line.”

With new technology, excuses are running out
Forty years ago, when the problem of cell line contamination and misidentification first came to light, the technology necessary to identify contamination was limited to karyotyping (11-13) and identifying biochemical polymorphisms based on isozyme expression. In the 1990s, DNA fingerprinting was introduced (14), but the research community made little use of it for identifying cell line contamination due to the required cost and the effort. “I believe widespread adoption of DNA fingerprinting by cell line stock centers is one approach,” says Badge, “but this needs to be combined with either practical assays that individual researchers can use to validate their own cell lines, or ready access to cheap, robust, and certified authentication services.”

According to Capes-Davis, biological researchers could benefit from the standards and protocols for authenticating cell lines developed by other scientific communities (15). “It’s relatively easy these days to detect contaminated human cell lines, thanks to the work done on identification within the forensic community,” she says. “The accepted method they use is short tandem repeat (STR) profiling.” A PCR-based method, data is interpreted to produce a numerical code, which can then be compared to a database of other STR profiles. This makes it possible for a laboratory to compare their cell line sample to one contained in the reputable STR cell bank; authentication is ensured when the sample matches.

Protocols are technologically feasible. But is that enough to get researchers to authenticate their cell lines? According to Roland Nardone, professor emeritus at Catholic University in Washington, DC, it isn’t.

Enforcing compliance
Nardone made waves by publishing a treatise on the topic in 2007: “Eradication of cross-contaminated cell lines: a call for action” (16). In this paper, along with a 2008 peer-reviewed follow-up in BioTechniques (17), Nardone proposes that granting agencies and journals enact a zero-tolerance policy that would limit researchers’ access to funding and publication if they do not authenticate their cell lines.

Though the proposal has been met with resistance, it may be the best way to achieve universal compliance, according to Badge. “The most reasonable way to have these authentication requirements enforced is to make it a condition of publication.”

As the largest funding agency in the United States, the NIH is in a unique position to take the lead in requiring the research community to authenticate their cell lines before they commence with a research project. So far, the NIH has issued a notice (NOT-OD-08-019) encouraging cell line authentication, but authentication is not a requirement for receiving an NIH grant.

How would researchers cope with a crack down?
According to Tetsu, STR analysis is most likely to be adopted for widespread use if cell line authentication became mandatory. “Publishers should ask researchers to perform STR analysis before they submit a paper,” he says, who also stressed the need for a complete database of all cell lines’ STR profiles. “The NIH should be responsible for creating such a database. In addition, we should take action to request authors and publishers to retract old papers if they used contaminated cells in the past.”

“The ATCC-SDO is currently writing a standard for testing human cell lines by STR profiling,” said Capes-Davis. “This will give help for laboratories setting up testing, or wanting background on the test even if they choose to send their samples away for external analysis.” The ATCC-SDO standard will give some researchers the means to hold themselves accountable.

STR analysis and authenticated data banks will make it easier for researchers to comply with new standards, but scientists are still developing even better technologies to identify contamination. “My group just submitted a paper on a system that enables genetic identification of mouse cell lines using strain specific retrotransposon insertions,” says Badge.

All scientists that spoke with BioTechniques were in agreement that the best way to eliminate contamination is to require researchers to authenticate; journals and funding agencies are in the right position to enforce this. But regulatory groups need to make validation easier and more beneficial for researchers and help them transition to checking cell line authenticity regularly.

Whether by being active in groups like the ATCC-SDO like Capes-Davis, by publishing data on contaminated lines like Tetsu, or by investigating new authentication technologies like Badge, the driving force behind efforts to reduce cell line contamination comes from biological researchers themselves. And these researchers agree that change is due in the overall community’s approach to cell line contamination. What remains to be seen is whether advocates will generate enough uproar to make agencies like the NIH or peer-reviewed journals force authentication upon those who do not comply on their own, or whether researchers will begin holding themselves accountable.

References

  1. Gey, G.O., W.D. Coffman, and M.T. Kubicek. 1952. Tissue culture studies on the proliferative capacity of cervical carcinoma and normal epithelium. Cancer Research. 12:264–265.
  2. Gartler, S. M. 1967. Genetic markers as tracers in cell culture. Journal of the National Cancer Institute Monographs. 26:167–195.
  3. Nelson-Rees, W.A., R.R. Flandermeyer, and P.K. Hawthorne. 1974. Branded marker chromosomes as indicators of intraspecies cellular contamination. Science 184:1093–1096.
  4. Lucy, B. P., W.A. Nelson-Rees, and G.M. Hutchins. 2009. Henrietta Lacks, HeLa cells, and cell culture contamination. Archives of Pathology and Laboratory Medicine. 133:1463–1467.
  5. Dirks, W. G., H.G. Drexler, and R.A. MacLeod. 1999. F. ECV304 (endothelial) is really T24 bladder carcinoma: cell line cross contamination at source. In Vitro Cellular and Developmental Biology. 35:558–559.
  6. vanBokhoven, A., M. Varella-Garcia, C. Korch, and G.J. Miller. 2001. TSU-Pr1 and JCA-1 cells are derivatives of T24 bladder carcinoma cells and are not of prostatic origin. Cancer Research 61:6340–6344.
  7. Liscovitch, M., and D.A. Ravid. 2006. Case study in misidentification of cancer cell lines: MCF-7/AdrR cells re-designated NCI/ADR-RES are derived from OVCAR-8 human ovarian carcinoma cells. Cancer Letters 245:350–352.
  8. Phuchareon, J., Y. Ohta, J.M. Woo, D.W. Eisele, and O. Tetsu. 2009. Genetic profiling reveals cross-contamination and misidentification of six Adenoid Cystic Carcinoma Cell Lines: ACC2, ACC3, ACCM, ACCNS, ACCS, and CAC2. PLoS ONE 4:6.
  9. Rahbari, R., T. Sheahan, V. Modes, P. Collier, C. Macfarlane, and R.M. Badge. 2009. A novel L1 retrotransposon marker for HeLa cell line identification. BioTechniques 46:277–284.
  10. Capes-Davis, A., G. Theodosopoulos, I. Atkin, H. Drexler, A. Kohara, R.A.F. MacLeod, J.R. Masters, Y. Nakamura, Y.A. Reid, R.R. Reddel, R.I. Freshney. 2010. Check your cultures! A list of cross-contaminated or misidentified cell lines. International Journal of Cancer. 127:1–8.
  11. Rothfels, K.H., A.A. Axelrad, L. Siminovitch, E.A. McCulloch, R.C. Parker. 1958. Proceedings of the third Canadian Cancer Conference. 189–214.
  12. Defendi, V., R.E. Billimgham, W.K. Silvers, and P. Moorhead. 1960. Immunological and karyological criteria for identification of cell lines. Journal of the National Cancer Institute. 25:359–385.
  13. Brand, K.G., and J.T. Syverton. Results of species-specific hemagglutination test on transformed, non-transformed, and primary cell cultures. 1962. Journal of the National Cancer Institute. 28:147–157.
  14. Gilbert, D.A., Y. Reid. M.H. Gail, D. Pee, C. White, R.J. Hay, S.J. O’Brien. Application of DNA fingerprints for cell-line individualization. 1990. American Journal of Human Genetics. 47:499–514.
  15. American Type Culture Collection Standards Development Organization Workgroup ASN-0002. 2010. Cell line misidentification: the beginning of the end. Nature Reviews 10:441–448.
  16. Nardone, R.M. 2007. Eradication of cross-contaminated cell lines: a call for action. Cell Biology and Toxicology 23:367–372.
  17. Nardone, R.M. 2008. Curbing rampant cross-contamination and misidentification of cell lines. BioTechniques 45:221–227.

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2010 marked the beginning of our Methods-specific Newsletter series. Covering cell culture, microscopy, PCR, and antibody technology, BioTechniques brought you the latest methodological and technical advances in these exciting fields through weekly feature articles and news stories. If you enjoyed the Top Cell Culture Feature of 2010, check out the rest of the editors’ picks of our favorite methods-specific news features from 2010 here.