Cell Line Authentication Testing

Human cultured cell lines are used in a number of biomedical research and clinical applications, including cancer research, drug discovery, genetics and biobanking. However, misidentified human and animal cell lines have continued to be used even today despite multiple and repeated warnings, articles and letters by prominent scientists in the field calling for authentication.

The need is great for researchers to authenticate their human cell lines. The call for authentication has been made for over five decades now, yet still, many researches do not perform this vital quality assessment. Recently there have been more spotlights shining on this deficiency from National Public Radio (NPR) series last December, comments from Dr. Francis Collins of the NIH, Nature announcing changes to abstract submission and most recently, newly revised NIH grant application instructions. As a result, more institutions are coming around to offering, even requiring, human cell line authentication through their DNA core labs or service providers.

Cell line authentication is achieved by genetic profiling using polymorphic short tandem repeat (STR) loci. STR loci consist of repetitive sequence elements 3–7 base pairs in length. These highly discriminative markers can be utilized using rapid and inexpensive multiplex-PCR based method for the identification and detection of contaminating human cells.

Standards for Cell Line Authentications

To standardize STR analysis for human cell line authentication, the American Tissue Culture Collection (ATCC) Standards Development Organization Workgroup published ASN-0002-2011, which recommends the use of at least eight STR loci (TH01, TPOX, vWA, CSF1PO, D16S539, D7S820, D13S317 and D5S818) plus Amelogenin for gender identification for human cell line authentication. This comprehensive standard provides detailed information on preparing samples for analysis, general instructions for analyzing the PCR products using capillary electrophoresis, tips and recommendations on how to view and interpret the resulting DNA profiles and more.

1. Send cells or extracted DNA to an experienced STR profile testing service.
2. Compare the STR profile to a reference sample and other known cell line STR profiles.
3. Check the cell line name against the database of misidentified cell line.

Be sure to authenticate at key intervals in a project when you:

1. Establish or acquire a new cell line.
• At an early passage (within the first week of culture).
2. Start a new series of experiments.
3. Are routinely passaging cell lines.
4. See inconsistent cell behavior or unexpected results.
• Limit passage number because cell lines mutate over time.
5. Prepare to publish.
6. Freeze cell stocks to verify purity.

What are STRs?

Short tandem repeats or STRs are repetitive sequence elements 3–7 base pairs long scattered throughout the human genome. By amplifying and analyzing these polymorphic loci, then comparing the resulting STR profile to that of a reference sample, the origin of biological samples such as cells or tissues can be identified and verified. The more loci that are amplified, the higher the statistical power of discrimination due to the repeat variation among humans and the human cell lines derived from individuals.

STR Analysis of Cell Lines

The user is sent a simple text-based table and may also receive a copy of the electropherogram results from the STR analysis. The nomenclature is based on number of STR repeats for each loci tested.

Table 1: Example of text-based results

Figure 1. Example electropherogram results.