Abstract
The history of cell line misidentification goes back to more than 50 years ago when the first human cell line, HeLa, was established. HeLa, a glandular cervical cancer cell line established in 1952 in George Gey's laboratory at Johns Hopkins University, was known to contaminate many cell lines, due, in part, to primitive culture conditions. Even though techniques and culture conditions have improved, the problem of misidentified cell lines or cellular cross-contamination remains at a very high level. One-third of all human cell lines are thought to be misidentified. Recently, this information and more was highlighted in a Wall Street Journal article, “Lab Mistakes Hobble Cancer Studies but Scientists Slow to Take Remedies” 1 and in response, Biopreservation and Biobanking invited Yvonne Reid, Ph.D., of the American Type Culture Collection (ATCC) and Joe Mintzer, M.S.W., of the Coriell Institute to respond to the article and provide a more in-depth look at cell authentication.
Yvonne, from your experience with cell authentication—and involvement in creating the standardized consensus protocol—could you give us an idea of the current state of cell line authentication?
One of the most important pieces of information that came out of the ATCC paper Standard ASN-0002 - Authentication of Human Cell Lines: Standardization of STR Profiling was that we are able to identify the minimum number of markers needed for human cell line identification using short tandem repeat (STR) analysis. Retrospective studies on 500 human cell lines obtained from several Cell Banks showed that a minimum of 8 core markers (D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO) were needed to uniquely identify human cell lines and for STR profile cell line comparison. This Standard was published in February 2012 by the ATCC Standard Development Organization (SDO) and is now available at American National Standard Institute (ANSI).
In addition we are working with the National Center for Biotechnology Information (NCBI) to set up a public database of STR profiles for human cell lines. Once available, all scientists will have the opportunity to submit their STR profiles into this database as either “verified” or “unverified.” Unverified are STR profiles that are input directly into the database without review. Verified entries are STR profiles (electropherograms) that have first been reviewed by the National Institute of Standards and Technology (NIST) prior to being submitted to the database. It is the hope that this database will allow us to identify unique cell lines and misidentified cells lines and, importantly, will allow us to trace the source of misidentified cell lines. The ATCC Working Group strongly recommended that a scientist who develops cell lines obtains a baseline STR profile from the original tissue. Then, in the event that the cell line becomes misidentified there is some original information from the source material.
What solutions will advance cell authentication for biobanks?
Cell authentication encompasses several methods, including correct morphology, optimal growth condition, correct species, free of microbial contamination, and correct cell function. 2 But one of the single most important authentication methods is to determine the unique identity of the cells being used. This can be done simply by using STR analysis. STRs are short tandem repetitive sequences that are distributed throughout the genome and are among some of the most informative markers for human cell identity. A panel of 17 markers plus amelogenin (which is used for gender determination) is capable of distinguishing among human cells at 1×10−18.
I recently had the opportunity to co-chair the Working Group that wrote the ATCC paper, Standard ASN-0002 - Authentication of Human Cell Lines: Standardization of STR Profiling. The solutions that were recommended for advancing the authentication of cell lines can certainly be applied to biobanks. The authentication method recommended was STR analysis for the following reasons:
• accessible, • easy to perform, • affordable, and • robustness.
We certainly think that STR analysis is the most suitable method at this time for the authentication of human cell lines. 3
What are some of the pitfalls and problems with this analysis?
STR analysis is used for intraspecies identification of human cell lines and tissues and is very useful in differentiating one human cell line from another at very high resolution. The probability of two unrelated human cell lines having an identical DNA profile using 17 STR markers is 1×10−18. However one concern of STR analysis is that it is not very useful in distinguishing between human cell lines derived from different tissues taken from the same individual. For example, a hepatocyte cell line derived from the liver of a patient will have the same STR profile as a colon cell line derived from the same individual.
Also, the STR markers are human-specific and are not able to detect other species.
Yet another drawback is the interpretation of the data. Most human cell lines have aneuploid karyotypes which often give rise to chromosome duplications, mutations and rearrangements. These chromosomal abnormalities may be the source of complex STR profiles that may be difficult to interpret. This PCR-based assay can have artifacts such as stutter peaks, allele drop outs and off-ladder alleles that may interfere with interpretation of the data.
What is still needed in order for authentication to be widely accepted?
Unfortunately, there is a certain amount of apathy among scientists about accepting that cellular cross-contamination or misidentification of human cell lines is a problem. This problem is pervasive within the scientific community. It is estimated that about one third of all cell lines used in research are misidentified.
As stakeholders we all have a responsibility in trying to resolve this problem. As researchers, we have the responsibility of confirming, often, the identity of our cell lines or tissues used for research; as scientific societies, we have a responsibility to educate our audience about the issues and consequences of cell line misidentification; funding agencies should strongly consider the implications of funding programs that lack proper authentication protocols; editors of journals have the responsibility to mandate cell authentication as a pre-requisite for publication. Finally, the public has the responsibility to hold us all responsible.
What is the future direction of authentication?
My hope is that scientists will no longer ‘bury their heads in the sand’ and will take full responsibility for their actions. Bad science hurts all of us. Boonstra 4 and his colleagues in The Netherlands showed in a recently published paper that the misidentification of three of the most frequently used esophageal adenocarcinoma cell lines (SEG-1, BIC-1, SK-GT-5) were misidentified and found to be lung, colon, and stomach. The information obtained from these cell lines led to over 100 scientific publications, 11 U.S. patents, at least 3 grants and recruiting patients for clinical trials had begun. This is an indirect way to say that I hope that publications such as this article will be a wake-up call to all of us and we take heed and begin to authenticate our cells more often—“garbage in, garbage out.”
Are there any quality or performance programs that laboratories participate in (or should participate in) if they are performing this service?
After the standards paper on cell line authentication was completed, the Working Group was committed to ensuring that the issue of cell line misidentification was not forgotten. Members of the group, which has representation from industry, academia, government agencies, and cell banks, established a committee which now focuses on continuing to address the issue of misidentified cell lines.
Are there international societies or congresses that are entirely focused on authentication?
I am not aware of scientific societies that focus entirely on authentication of cell lines. However, there are several societies, such as International Society of Biological and Environmental Repositories (ISBER) and the Society for In Vitro Biology (SIVB), which focus on best practices for repositories and tissue culture, respectively.