February 1, 2014 (Vol. 34, No. 3)
John Masters, Ph.D. Professor University College of London
Yvonne Reid, Ph.D. Manager, Scientist ATCC
Standards Can Ensure Responsibility and Reproducibility
Failure to use biological standards or standardized methods—such as the validation of antibodies, application of biomarkers, reporting of OMICs studies and clinical trials, and the quantitation of gels, etc.—and their consequences can be found systemically throughout the life sciences. Recently, the critical nature of these issues was highlighted by the Global Biological Standards Institute (GBSI) in their report, “The Case for Standards in Life Science Research: Seizing Opportunities at a Time of Critical Need” at www.gbsi.org/CaseforStandards.
This report, which was based on an extensive research program composed of broad secondary research of over 100 peer-reviewed references and a primary interview process consisting of almost 60 individual discussions, emphasized the need for standards to counteract the detrimental effects of irreproducibility. In this article, we will examine one of the examples highlighted in the GBSI report—the misidentification of human cell lines—and show how the introduction of a single consensus standard on cell-line authentication has since transformed scientific attitudes and practices.
The misidentification of cell lines is a problem that has affected the life sciences enterprise almost since the very first human cell line, HeLa, was derived from the cervical cancer cells of Henrietta Lacks in 1951 by George Gey, Johns Hopkins University. As this scientific achievement offered profound benefits to the scientific community, George Gey distributed HeLa cells worldwide over the next few years. Shortly thereafter, there was acceleration in the availability of immortal human cell lines developed from cancerous and normal tissues, despite the fact that it had taken thousands of attempts to grow the first human cancer cell line. Unfortunately, it soon became obvious that many of these newly derived human cell cultures were contaminated with HeLa cells.
It was nearly 50 years ago in 1967 that Dr. Stanley Gartler, now professor of genome sciences, University of Washington School of Medicine, demonstrated the problem of cell-line misidentification by using isoenzymology. He showed that the same Phospho-glucomutase PGM-1 locus and a fast variant of Glucose-6-phosphate dehydrogenase-A (G6PD-A) G-6-PD, observed almost exclusively in African Americans, were expressed in the all the human cell lines he tested. However, some of the cell lines he examined were reported to be Caucasian in origin.
The findings indicated that the cell lines were contaminated with HeLa. Regrettably, Dr. Gartler’s disclosure was not supported and appreciated by the cell culture community. In fact, over the years, many scientists have deliberately ignored this evidence and continue to use the misidentified cell lines described by Dr. Gartler.
The full extent of this pervasive issue was further revealed when Dr. Walter Nelson-Rees, who managed the cell bank at the Naval Biological Laboratories, began an initiative to eradicate the use of misidentified cell lines. Through karyotyping, he demonstrated that many human cell lines were in fact HeLa cells.
In 2006, the cell culture scientist, Dr. Roland Nardone, professor emeritus, Catholic University, Washington, DC, took over where Dr. Nelson-Rees left off and started another campaign against cell-line misidentification. Progress was difficult for many reasons, including the fact that the initiative was being driven by a single concerned scientist. It became clear that another approach was needed.
It has been estimated that approximately 15% to 30% of all cell lines used in basic and applied research are misidentified. This misuse had led to false data, loss of time, and an inefficient use of public and private funding on research that may be flawed or misleading, and may have encouraged other scientists to use the false cell lines inappropriately. In one example, misidentified esophageal adenocarcinoma cell lines have led to patent applications, NIH grant funding, and recruitment of patients for clinical trials. Over the years, this problem has mushroomed to over 400 examples of false cell lines.
How could individual scientists get their voices heard and begin solving this problem? The answer came through the publication of a single consensus standard on the authentication of human cell lines.
In 2009, a group of concerned scientists approached the ATCC Standard Development Organization to establish a consensus standard on the authentication of human cell lines by STR profiling. The working group, made up of scientists from academia, industry, government, and cell banks, wanted a written standard that was robust, consistent, inexpensive, and easily available to all scientists in the life sciences. In January 2012, the standard was published by the American National Standard Institute—ASN-0002 Authentication of Human Cell Lines: Standardization of STR Profiling.
We chaired the international working group, which consisted of 22 members and alternates from five countries. The members of the working group were very dedicated and committed to the completion of the standard; they gave their time freely until the consensus standard, a document of more than 100 pages, was published in 2012.
The publication of ASN-0002 Authentication of Human Cell Lines: Standardization of STR Profiling has since transformed attitudes. While the problem of misidentified cell lines has not yet disappeared, there has been a remarkable change in the attitude of the scientific establishment.
For example, several testing laboratories are now offering services for authenticating human cell lines, using the standard as the acceptable protocol. Further, some journals are now demanding that all cell lines described in publications must be authenticated as a prerequisite for publication.
Such pioneers include the International Journal of Cancer and Cancer Research (and other journals published by AACR). Nature has published a letter about the standard and is developing guidelines for authors concerning authentication. In fact, one of the Nature journals recently retracted a paper because the cell line used was false. Moreover, some funding bodies are now suggesting that cell lines to be used in grants should be checked for authenticity.
Overall, more has been achieved during the short period since the standard was published than had been achieved in the whole of the previous 50 years by individual scientists. The power of standardization to change policy and engender understanding of a problem is evident and needs to be extended to all aspects of the life sciences.