Last week, we finished our Ask the Expert Discussion on Cell Line Authentication. Inadvertent cell line contamination is a serious concern for many researchers.  Failure to monitor cell lines for contamination may result in compromised data.  Current estimates are that 18% to 36% of all active cell lines are either cross-contaminated or misidentified.1  This level of concern has led a number of journals to actively encourage cell line authentication prior to submission for publication. Cell line authentication testing is a good quality control step for any laboratory utilizing human cell lines and/or stem cell lines. These were the topics covered during our Ask the Expert session “The Role of Cell Line Authentication in Today’s Biomedical Research World.”

Hosting this week’s session is Erin M. Hall, M.S., Director, Cell Line Authentication Services, Genetica DNA Laboratories, LabCorp Specialty Testing Group. Erin received a master’s degree in Forensic Science from Pace University in New York City and a bachelor’s degree in Molecular Biology from the College of Mount Saint Joseph in Cincinnati, Ohio. Prior to joining Genetica, Erin worked as a laboratory manager and researcher in the Pharmacology department at Cornell University’s Weill Cornell Medical College. She designed and executed complex experiments that examined the effects of environmental toxins on liver enzyme production utilizing HPLC, UV/vis spectroscopy, Western blot, and PCR analysis. Her work contributed to several published journal papers (under Erin Labitzke), most recently including being cited as first author on a paper related to enzymes present in mitochondria.

Question topics included:

• Implementing cell line authentication in labs
• Karyotyping and isoenzyme analysis
• STR DNA profiles
• Cell line misidentification and cross-contamination
• Journals requiring cell line authentication prior to publication
• Regulatory requirements

I have selected a few of the submitted questions and answers to include below. For a full list of questions and answers, please see Ask the Expert – Cell Line Authentication.

How does cell line misidentification and cross-contamination happen in the lab?

The Answer:

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.

I perform Karyotyping and isoenzyme analysis in my lab, why is STR DNA profiling a better technology than other methods for authentication?

The Answer:

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.

How do you recommend companies implement cell line authentication? At what point in the process would you get your lines tested and what are the issues that can happen if you choose not to test your cell lines? Thanks

The Answer:

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).

For more information about the topic of cell line authentication, please also see Erin’s previous guest blog “Has your research been compromised? The role of cell line authentication.”