Reproducibility: Changing the Policies and Culture of Cell Line Authentication
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COMMENTARY Reproducibility: changing the policies and culture of cell line authentication Leonard P Freedman1, Mark C Gibson1, Stephen P Ethier2, Howard R Soule3, Richard M Neve4 & Yvonne A Reid5 Quality control of cell lines used in biomedical research is essential to ensure reproducibility. Although cell line authentication has been widely recommended for many years, misidentification, including cross- contamination, remains a serious problem. We outline a multi-stakeholder, incremental approach and policy-related recommendations to facilitate change in the culture of cell line authentication. Advances in life science research build drug target discovery and to generate diag- The Global Biological Standards upon the reproducibility of previously nostic hypotheses in multiple areas of bio- Institute (GBSI) formed the Cancer Cell published data and findings, yet irrepro- medical research9. In these uses, accurate Authentication and Standards Task Force ducibility in basic and preclinical biologi- documentation of species, sex and tissue in 2014 to (i) identify and overcome exist- cal research is a pervasive, expensive and of origin is integral to interpretation and ing barriers to the use of currently available increasingly well-recognized problem1,2. validity of research results. It is also impor- cell authentication tools and (ii) support Also called replication, validation, verifica- tant to monitor genotypic or phenotypic the development, evaluation and applica- tion or reanalysis3, in simplest terms, repro- changes (i.e., drift) that might occur over tion of policies, novel technologies, and ducibility means that an experiment should time10. standards for expanded cell authentica- be able to be confirmed in an independent Correct identification of the origin of a tion. Cell line misidentification and micro- laboratory with results that broadly support cell line is simple. Cell line authentication bial contamination are not new problems, Nature America, Inc. All rights reserved. America, Inc. Nature the conclusions of the original scientist. can be achieved by determining the genetic and although several consistent solutions 5 Excluding deliberate scientific miscon- signature (by profiling or fingerprinting) involving authentication have been offered 4 © 201 duct , irreproducibility typically results and comparing it with established data- (and in some limited venues, adopted) over from errors or flaws in one or more of the bases to confirm identity11. From 2011 to the years16, such issues have proven sur- following areas of the research process: ref- 2012, an international group of scientists prisingly difficult to eradicate. This paper erence materials, study design, laboratory from multiple stakeholder groups collabo- reflects the deliberations and reports the npg protocols, and data analysis and report- rated to develop an accredited standard conclusions and recommendations of the ing5,6. Irreproducible preclinical research that describes optimal cell line authentica- Task Force to affect meaningful change in contributes to both delays and increased tion practices based on STR (short tandem both the policies and culture of cancer cell costs in drug discovery. repeat) profiling12. The International Cell line use and authentication across the entire One common contributor to irreproduc- Line Authentication Committee was formed biomedical research community. Although ibility in the life sciences is the widespread following publication of the STR profiling microbial contamination of culture systems use of misidentified (including by intraspe- standard to make cell line misidentification by bacteria (particularly mycoplasma17), cies and interspecies cross-contamination more visible and to promote awareness and fungi, viruses and other microorganisms and simple mislabeling) or microbially con- use of authentication testing. However, there continues to be highly problematic and taminated cell lines isolated from various is little evidence that authentication is widely expensive18, a detailed discussion of those human tissues7,8. Cell lines have been used used in the life sciences. Many researchers issues is beyond the scope of this paper. for decades to study basic biological mecha- are simply unaware of the need to establish nisms and serve as preclinical models for and carefully maintain cell cultures and tech- The problems niques, or they are aware but do nothing to Prevalence of misidentified cell lines. 1Global Biological Standards Institute, Washington, validate their cell lines. Several journals, Reports from major cell repositories and DC, USA. 2Hollings Cancer Center, Medical 13 University of South Carolina, Charleston, South including Nature , now require or strongly research laboratories indicate that a wide Carolina, USA. 3Prostate Cancer Foundation, recommend cell line authentication for stud- variety of cell lines submitted or tested Milken Institute, Santa Monica, California, USA. ies they publish. Yet, despite these efforts, are misidentified (Table 1). A widely cited 4 Department of Discovery Oncology, Genentech new reports of misidentified or contami- review examining the prevalence of this Inc., South San Francisco, California, USA. 5ATCC, Manassas, Virginia, USA. nated cell lines still appear in the scientific problem from 1968 to 2007 estimated that e-mail: [email protected] literature14,15. between 18% and 36% of cell lines might be NATURE METHODS | VOL.12 NO.6 | JUNE 2015 | 493 COMMENTARY Table 1 | Select reports of misidentified or cross-contaminated cell lines by major cell before they were found to be derived from repositories human ovarian carcinoma cells (now redes- 29 Total number Number of Percentage of ignated NCI/ADR-RES) . For perspective, Cell type of lines false cell lines false cell lines Ref. given the cost of an average US National Lymphoma, leukemia 550 82 15 39 Institutes of Health–funded breast cancer Ovarian cancer 51 15 30 40 research grant in 2013 ($370,000), as much Adenoid cystic carcinoma 6 6 100 41 as $100 million of research funding may Thyroid cancer 40 17 43 42 have been spent using this misidentified Head, neck cancer 122 37 30 43 cell line alone. Esophageal adenocarcinoma 14 3 21 44 The need for new cell authentication tech- Total 783 160 20 (average) niques. If we exclude ignorance and indif- ference, cost and simplicity of assays appear misidentified or cross-contaminated, with rials in the public domain will remain or to be the biggest roadblocks to universal use only a small improvement in rates over eventually become compromised. of cell authentication. The introduction of time19. A recent study reported that only cheap, commercially available assays that 43% of cell lines could be uniquely identi- Use and cost of misidentified cell lines. can be easily implemented and interpreted fied (i.e., an unambiguous name or iden- Despite the availability of affordable com- in any laboratory will greatly increase the tifier was provided as well as a source for mercial kits to profile cells and measure likelihood of universal adoption. Because the line such as a vendor or repository) by contamination, it is unknown how many STR profiling alone may never achieve their description in an evaluation of over laboratories authenticate or conduct qual- those goals, alternative methods are need- 200 biomedical papers20. Furthermore, ity control (QC) on their cell lines and how ed. these surveys do not reflect (i) cell lines often. A decade-old survey25 reported that Single-nucleotide polymorphisms that were incorrectly identified when they just one-third of laboratories tested their (SNPs) are genetic variations between were first cultured, (ii) cell lines that have cell lines for identity. A recent Nature Cell members of the same species. SNPs within been displaced (cross-contaminated) by Biology editorial reported that an audit of a specific locus are conserved during evo- unknown cell types, or (iii) cell lines that papers with data generated using cell lines lution and can be used as a genetic test of were mislabeled or contaminated in indi- published between August and December identity. SNP typing assays have been pub- vidual laboratories. For these reasons, the 2013 revealed that only 19% of published lished for forensic and cell line authentica- true incidence of misidentified cell lines is papers conducted (or at least reported con- tion applications30,31. 52-plex SNP assays probably even larger. ducting) cell line authentication26. appear to have the same rate of discrimina- Several organizations21,22 promote and HeLa is by far the most common con- tion as 16-plex STR, whereas smaller pan- 27 Nature America, Inc. All rights reserved. America, Inc. Nature recommend best practices for handling tributor to cross-contaminated cell lines . els of 24 SNPs are equivalent to 8-plex STR 5 biospecimens and biological resources, Another poster child for misidentified cell panels32. Commercial kits are available (for 23,24 © 201 including cell lines and cell banking , lines is MDA-MB-435. More than 1,000 example, iPLEX Pro Sample ID Panel from but none is recommended by the major- articles have been published on this cell line Agena Bioscience and SNP Trace Panel ity of key life science stakeholders. The as a triple-negative, metastatic breast cancer from Fluidigm), but adoption as a general expanded adoption of best practices cell line that grows well in vivo. It was later method for cell line authentication is not npg should, over time, lead to additional con- reported to have