Are we inseparable? Reproducible bioassays and authenticated cell lines: New initiatives by the Global Biological Standards Institute

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Are we inseparable? Reproducible bioassays and authenticated cell lines: New initiatives by the Global Biological Standards Institute

by Sherry Ward, AltTox Contributing Editor
Posted: May 29, 2015

The #authenticate Campaign to Improve Research Reproducibility

Analysis by the Global Biological Standards Institute (GBSI) indicates that hundreds of millions of dollars in US government research funding is being compromised due to the use of contaminated and/or misidentified cell lines.

Various researchers from academia and industry have recently committed to work together to address the long-known problem that many of the cell lines used in published research have not been sufficiently authenticated, and that more robust guidance and/or standards are needed to improve research reproducibility and ensure the best investment of our research dollars. You may have seen the recent commentaries by NIH leadership on NIH’s plan to address this topic (Lorsch, Collins & Lippincott-Schwartz, 2014; Collins & Tabak, 2014).

GBSI is currently conducting an important project to gather information about laboratory practices related to cell line authentication and antibody validation. To participate and include your experience/expertise as part of GBSI’s results, you can access one or both surveys at the link below.

GBSI survey:
This survey will close on Friday, June 5th at 5PM EST.

Researchers are also invited to read the #authenticate campaign press release, and sign the #authenticate pledge.

The involvement and role of GBSI will be further explained in sections below.


HeLa cells are the human cervical cancer cell line isolated in 1951, and chronicled in the book The Immortal Life of Henrietta Lacks (Skloot, 2011). HeLa cells are credited with being the first immortal human cell line to be grown in a laboratory. The cell line was commercialized in the 1950’s and widely distributed among biomedical researchers, leading to the recognized importance of HeLa cells in the development of present day cell culture techniques as well as their role in many research and medical breakthroughs.

The wide availability of the immortal HeLa cell line has been described as an enabler for scientists to replicate/reproduce experiments carried out in other labs (Skloot, 2011). On the other hand, HeLa cells are also infamous for having contaminated many other cell lines over the decades. A simple search in PubMed for “HeLa cell contamination” illustrates that cross-contamination of biological samples and cells has been widely detected (examples include: Buehring, Eby & Eby, 2004; Capes-Davis et al., 2010; Jager et al., 2013; Kniss & Summerfield, 2014; Nelson-Rees, Daniels & Flandermeyer, 1981). Masters (2002) explained that cell line contamination was recognized by the 1960s, and his article provides an historical overview of the issue.

Calls for cell-line authentication to prevent the publication of misleading and/or irreproducible results due to the use of contaminated cell lines have also appeared in the literature for many years (examples include: ATCC SDO, 2010; Hughes et al., 2007; Nardone, 2007). Lists of contaminated/misidentified cell lines are identified in various publications and databases (ATCC, accessed May 25, 2015; ICLAC, 2013; Capes-Davis et al., 2010).

While not all cell lines are immortal, even cell lines with a finite number of population doublings need to be maintained in a consistent and reproducible manner to ensure reproducible experimental results. Cell banking/bioresource organizations, such as the American Type Culture Collection (ATCC), have provided information to educate researchers on the importance of cell authentication and other good cell culture practices through the publication of standardized procedures. The ATCC Technical Bulletin Cell Line Authentication Test Recommendations (ATCC, 2010) defines the authentication of a cell line as “the sum of the process by which a line’s identity is verified and shown to be free of contamination from other cell lines and microbes.” The five fundamental tests ATCC recommends to ensure cell lines “are uncontaminated and correctly identified” are the following:

  • Microscopic cellular morphology to identify the state of the cells. Comparative photomicrographs can be used to document reproducible cellular morphology. Cell morphology can vary depending on a number of factors, including state of differentiation, cell confluence, or contamination.
  • Cell growth curves to evaluate baseline population doubling time and consistency of cell proliferation. Cell proliferation can slow when cells start to senesce or undergo other changes.
  • Isoenzyme analysis to verify the cell line’s species of origin. This testing can be conducted following a published protocol or can be contracted out to a testing laboratory.
  • Short tandem repeat (STR) analysis to establish a DNA fingerprint for human cell lines. Each human cell line has a unique STR profile that can be used to identify the cell line in subsequent analyses. Various testing laboratories can conduct this analysis.
  • Mycoplasma infection testing. This common problem in cell cultures can adversely affect experimental results. Reagents are available for routine testing, or samples can be sent to contract testing laboratories.

In 2007, ATCC formed the ATCC Standards Development Organization (ATCC SDO) “in response to the needs of our industry.” The purpose of SDO is “to assure the development of nationally and internationally accepted standards for biomaterials that meet International Organization for Standardization (ISO) guidelines for standards development, and are recognized by the American National Standards Institute (ANSI).” An example SDO’s accomplishments is the adoption of the ANSI guidance “Authentication of Human Cell Lines: Standardization of STR Profiling” (ANSI/ATCC ASN-0002-2011).

Various cell banking and research organizations formed the International Cell Line Authentication Committee (ICLAC) in 2012 “to make cell line misidentification more visible and to promote awareness and authentication testing as effective ways to combat it.” ICLAC has published a database of more than 400 cross-contaminated/misidentifed cell lines, and provides guidance to researchers on human cell line authentication practices.

Another organization seeking to expand awareness among researchers and research organizations on the importance of cell line authentication is the Global Biological Standards Institute (GBSI). Since its creation in 2013, GBSI has been actively working to advance “the use of best practices and standards to improve the quality of life science research.” Based upon initial work with a panel of experts to define the issues, they released their first report, The case for standards in life science research. In November 2014, GBSI organized The BioPolicy Summit in Washington, DC, where panel members discussed important questions such as whether grant-making programs and scientific journals should require cell line authentication. The BioPolicy Summit also led to several public education radio reports on NPR Morning Edition.

GBSI’s current initiatives include the #authenticate campaign and the stakeholder surveys described above.

Another promising initiative is their educational program. Freedman and Siegel (2015) explain that “Fixing the problem demands a systematic incremental approach with commitment by all stakeholders and that embraces the importance of targeted training and education.” Thus, as part of their educational program, GBSI plans to develop an online, interactive, multimedia training module on cell authentication that can be used as stand-alone training or as part of a class. The training will go beyond the static push mode of current webinars, even planning to track effectiveness at the level of laboratory implementation.

Concluding Thoughts and Questions

These issues seem to be particularly important to the field of in vitro toxicology, where reproducible results are the foundation of test method validation and the use of bioassay data for regulatory decision-making, and should be of particular concern to regulators who accept this data.

A few questions for our AltTox readers:

  • Do you find this topic relevant to the development and use of bioassays for toxicity testing?
  • Do you see a role for research funding organizations, journal editors, research institutions, government policy, and/or improved education as the means to promote better implementation of cell line authentication and research antibody validation practices?

In addition to participating in the GBSI initiatives, readers are encouraged to post a comment on AltTox (post below, or submit to for posting in the Community Blog), or to submit an expert commentary as a New Perspectives article. Questions on the procedures for participating in any of these interactive options can be sent to