The Role of Cell Line Authentication in Today’s Biomedical Research World

STR DNA profiling is a method of human identification that allows for determination that the cell line was derived from a specific human – no two humans have the same STR DNA profile except for identical twins. Typically, karyotyping and isoenzyme analysis do not distinguish individual humans.

Unfortunately, mistakes can happen in even the strictest of labs. The infamous HeLa cells, which are extremely aggressive and fast growing, are known for being able to contaminate other cultures via aerosol droplets. A simple mislabeling of a flask could cause misidentification of the cell line in use. Not following stringent cell culture rules is also known to perpetuate the problem; sharing media and other lab supplies amongst multiple cell lines could risk contamination. Also, sharing cell lines between laboratories could be fuelling the problem – you may have unknowingly received a misidentified cell line.

There are currently several well-known journal publishers that have guidelines for cell line authentication testing prior to submission including (see instructions to authors for all journals):
1. American Association for Cancer Research (AACR) Journals, which includes - Cancer Discovery, Cancer Epidemiology, Biomarkers and Prevention, Cancer Prevention Research, Cancer Research, Clinical Cancer Research, Molecular Cancer Research, Molecular Cancer Therapeutics
2. BioTechniques
3. Cell Biochemistry and Biophysics
4. In Vitro Cellular & Developmental Biology – Animal
5. International Journal of Cancer
6. Nature Publications

A) Companies looking to implement a cell line authentication program need to decide if they wish to perform the testing internally or reference the samples to a laboratory specializing in cell line authentication. To test internally, the technology currently recommended by ANSI-ATCC standards is STR DNA analysis. This would require purchasing a genetic analyzer, thermal cyclers, and other supporting equipment. The laboratory would also need fragment analysis software and training to interpret the STR profiles and mixtures, as well as, training on identifying and troubleshooting anomalies. The commercial kits available for STR profiling should be validated at a minimum for reproducibility and sensitivity. Choice of kit would also be a consideration. Mixtures, which are common in cell line authentication testing, should also be validated as part of the setup. Some guidance on running and interpreting STR profiles can be found at ATCC SDO, Authentication of Human Cell Lines: Standardization of STR Profiling ATCC SDO document ASN-0002. Manassas, VA: ATCC Standards Development Organization, 2011.

Outsourcing the samples has the advantage of no capital expenses, no validation studies or training needed, and the reference laboratory should be able to generate appropriate reports. The company seeking a provider can find a number of qualified laboratories on the internet. Accreditation by an organization with standards for STR DNA profiling methods may also be desirable. Examples of accreditation agencies would be AABB, the College of American Pathologists (CAP), ISO (usually ISO 17025 or 15189) and various forensic organizations.

B) You should consider performing cell line authentication when a cell line is acquired, before starting a new series of experiments, when results using the same cell line are inconsistent or unexpected, to establish a STR DNA profile for a new established cell line, prior to freezing cell stocks for future use and every 2-3 passages during active growth. Ideally, for new “in-house” developed cell lines you would compare a low passage number of the established cell line to the original patient from which the cell line was derived (blood or solid tumor sample); this sets the “reference” profile for all future authentication testing with that cell line. If you plan to publish, checking with the journal you would like to publish in for their requirements, if any, may be prudent.

C) Failure to test your cell lines for authenticity could lead to compromised data – it is estimated that up to 40% of all published peer reviewed papers are affected by cell line misidentification and/or cross-contamination (Chatterjee, Science, 2007). There have been retractions of journal articles due to this problem – one example is the 2005 discovery that stem cells could seed cancer (Garcia-Castro, J. et al, Can Res, 2005). In 2010 this article was retracted because the authors were unable to reproduce some of the reported spontaneous transformation events; a contamination with the human cancer cell line HT-1080 was identified as the source of the problem (Garcia, S. et al, Exp Cell Res, 2010).

The STR DNA profile can determine if there is no contamination or alteration of the cultured cell line as compared to the original cells. The results may be able to identify a contaminant and also measure instability or other changes in the cell line. There are several mathematical measures that can assist in the probability of a match when compared to the reference cell line (see previous question/answer for reference to ANSI/ATCC ASN-0002 guidance document).

There are no specific regulations for cell line testing. See below for information on characterizing cell lines.

US FDA – “Points to consider in the characterization of cell lines used to produce biologicals. Department of Health and Human Services, Center for Biologics Evaluation and Research, 1993”.

For information on cell line authentication requirements for journal publications, please refer to the individual instructions to authors for each respective journal. See previous question/answer in this session for a list of journals that have guidelines for cell line authentication testing.