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Working together to eradicate cell line misidentification and contamination: New practices and policy
One of the most serious issues facing the biomedical research community today is the authentication of human cell lines used in research and drug development as models of normal and cancer tissue. Cell lines constitute an important scientific resource, enabling investigators to unravel cancer mechanisms that were once intractable to our methods, and to screen more potential drug candidates than ever before. Progress is being undermined, however, by cell line misidentification and cross-contamination. In this article, we will address the history of the problem, its implications to both cancer research and drug discovery and the options available to investigators concerned with validating their materials. Lastly, we will highlight how a new ANSI consensus standard and efforts by granting agencies and journals to require cell line authentication as a condition for funding and publication, and offer hope for a future free from the plagues of misidentified and contaminated cells.
Cell line misidentification and contamination is not a new problem. In the 1970s, Walter Nelson-Rees, who then ran the Berkeley cell bank for the National Cancer Institute, used karyotyping to determine that cell lines submitted to the bank were cross-contaminated by HeLa cells. Nelson-Rees embarked on a crusade against contaminated cell lines, which culminated in a 1981 Science paper listing publications that had used the flawed material. His gambit was met with outrage from authors of the implicated work, and his crusade faltered. In 2004, Roland Nardone, then a professor at Catholic University, picked up Nelson-Rees' mantle and championed efforts to raise awareness of the scope of the cell line misidentification problem. Nardone has emphasized the need for training in cell authentication to be added to conference agendas and is still garnering the support of professional societies and funding agencies to require cell line authentication.
Despite these renewed efforts, the consequences of using unauthenticated cell lines are already being brought to bear on researchers. For example, in 2010, a 2005 Cancer Research article that suggested human adult stem cells were prone to spontaneous transformation in vitro1 was retracted because the phenomenon was actually due to contaminating immortalized cells2. A year later, researchers hoping to develop therapeutic tools for adenoid-cystic carcinoma had their paper retracted when it was found that the cell line used to perform the experiments was actually derived from cervical cancer3. Importantly, these papers were published in well-respected journals, which increase the likelihood that other investigators based their research on the flawed data and compounded the original error.
A handful of cases have been uncovered, but it is clear that countless more exist. A recent review of cell lines used to study esophageal adenocarcinoma found that many were actually derived from lung or gastric cancers. Data obtained through the use of these cell lines have been used to support clinical trials, grant applications, U.S. patents and publications4. Similarly, genomic profiling of 51 supposedly independent ovarian cancer cell lines revealed considerable redundancy and that several were actually derived from cervical cancer5. These studies refer to clinically relevant cell lines that are being used to design and test new drugs for cancer and other diseases, and suggest that patients are potentially being recruited to flawed drug trials. If we assume that the findings from these two studies are applicable across all clinical fields, then the number of advances that stand to be reversed is staggering.
An important first step towards remediation is to address the root cause and develop a plan for going forward. In 2004, a survey of 485 investigators indicated that only 15 percent of investigators relied on DNA- based assays to authenticate their cell lines6. Then, and most certainly today, many investigators rely on phenotypic traits to confirm cell identity. This is an unreliable method for confirming cellular identity, since phenotypic traits may change unpredictably as passage number increases7. Additionally, many laboratories make use of multiple cell lines or feeder cell co-cultures, which boost the probability that a cross-contaminating event will occur, and make reliable methods of cell authentication, such as those discussed below, even more important to rule out both intra- and interspecies contamination.
The gold standard in genomic-based methods of cell authentication is short tandem repeat (STR) profiling. STR profiling is a PCR-based approach that can discriminate the origin of the cell line down to the original donor. This technique takes advantage of variable microsatellite regions within the genomic DNA. Primers are designed to amplify these short repetitive sequences and the resulting amplicons are used to establish a profile. The profile acts as a genetic fingerprint that can be compared to a reference database to confirm the identity of the cell line7. STR profiling can be performed rapidly using commercial kits and services, which makes it an approachable option for all investigators. Additionally, because the microsatellite regions are defined, cell line profiles will be comparable between labs and easily collected into a centralized public database.
Although STR profiling is a high-resolution approach to confirming the identity of a cell line, it does have some limitations. First, primers to detect STR from non-human species are limited, and currently this method is not capable of identifying interspecies cross-contamination. Second, donor information and tissue samples for older, commonly used cell lines may not be available, therefore it can be difficult, if not impossible to develop reference standards for older lines8. In rare situations, more than one method may need to be used to fully validate the identity of a particular cell line. For example, karyotyping, isoenzyme analysis and human leukocyte antigen typing can be used to compliment STR profiling, and this may be necessary if the cell line is being grown on a mouse feeder cell layer or if the original cell stock has been lost.
The commercial availability of STR kits and authentication services make it easier for investigators to authenticate cell lines. However, the problem reaches so deep into the scientific community that institutional support and the international collaboration of subject matter experts is necessary to eradicate the use of unauthenticated cell lines. Responding to the recognized problem of cell line misidentification and the need for standardized methods that can be used to test cell lines early and often, a standard consensus method for cell authentication was released earlier this year by the ATCC Standards Development Organization (SDO) as ANSI Standard ASN-0002, "Authentication of Human Cell Lines: Standardization of STR Profiling."9 The ANSI standard recommends that cell lines be tested when a cell line is first grown in a laboratory, when preparing the initial frozen cell stock, after two or three passages while cells are expanded during the course of experimentation and at the time the research using the cell samples is published. The most important aspects of the standard are the discussions on the numbers and types of loci to be evaluated, quality control of the data, interpretation of the results (matching criteria, loss of alleles, etc.) and implementation of an STR database.
An international workgroup of scientists representing academia, regulatory agencies, major cell repositories, government agencies and industry, chaired by John R. W. Masters of University College London and Yvonne A. Reid of ATCC, worked together to develop the standard. The standard represents a collective experience and expertise that led to a refinement and consolidation of methods that should be of critical value to investigators who are working with human cell lines. Standardization fosters the reproducibility and comparability of research and development employing human cells, leading to a marked decrease in the misidentification and contamination of human cells used by the scientific community.
Scientific journals and funding agencies are called upon to require proof that all human cell lines used in research studies have been properly authenticated. Professional scientific societies such as ASCB and AACR are encouraged to sponsor conferences, workshops, webinars and training activities to facilitate the adoption of cell line authentication standards. The quality and validity of funded and published research will markedly improve as a result of the reduction in the use of misidentified and contaminated human cell lines.
Elizabeth Kerrigan is the director of standards, and Dr. Carolyn Peluso is a cell biology specialist, both at ATCC, a private, nonprofit biological resource center and research organization whose mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms, cell lines and other materials for research in the life sciences.
1. Rubin, L. and Haston, K.: "Stem cell biology and drug discovery," BMC Biology.
2. de la Fuente, R., et al.: "Retraction: Spontaneous human adult stem cell transformation," Cancer Research.
3. Retraction notice to "Nef from SIVmac239 decreases proliferation and migration of adenoid-cystic carcinoma cells and inhibits angiogenesis," Oral Oncology.
4. Boonstra, J., et al.: "Verification and unmasking of widely used human esophageal adenocarcinoma cell lines," Journal of the National Cancer Institute.
5. Korch, C., et al.: "DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination," Gynecology Oncology.
6. Buehring, G., Eby, E. and Eby, M.: "Cell line cross-contamination: how aware are Mammalian cell culturists of the problem and how to monitor it?" In Vitro Cell Development Biology Animal.
7. Kerrigan, L. and Nims, R.; "Authentication of human cell-based products: the role of a new consensus standard," Regenerative Medicine.
8. "Cell line misidentification: the beginning of the end," Nature Reviews Cancer.
9. ANSI/ATCC ASN-0002-2011: "Authentication of Human Cell Lines: Standardization of STR Profiling."