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Human Cell Line Authentication

Cross-contamination or misidentification of cell cultures has been a problem in biomedical research labs for a long time. In many cases, certain cell lines have been used in a lab for years without ever checking its authenticity or the possibility of cross-contamination. Everyone is aware that many cell lines are suspected to be contaminated with Hela cells.

Microbial contamination is another problem associated with cell cultures. Some are evident under the microscope unless it’s at a very low level. Mycoplasma, not seen under light microscope, is hard to detect and can have harmful effects on various cellular functions.

It is important to ensure that a cell line is not contaminated and is correctly identified before initiating research with a long-established cell line. This can avoid the generation of flawed and irreproducible results and the associated wastage of valuable time and resources.

One should also check the database of misidentified and cross-contaminated cell lines (http://iclac.org/databases/cross-contaminations/).

The best practice is to do the STR profiling (see below) of a cell line at the beginning of a project and check it periodically throughout the duration of the project.

Description of the method to authenticate cell lines is a requirement for NIH grant applications. Many journals such as Nature request authors to authenticate their cell lines prior to publication. Detailed information on culture methods, cell line authentication and mycoplasma testing are to be provided in the supplemental sections of many journals.

STR or short tandem repeat analysis is an effective method to ensure the validity of cell lines. The GenePrint® 10 System from Promega allows co-amplification and three-color detection of ten human loci (TH01, TPOX, vWA, Amelogenin, CSF1PO, D16S539, D7S820, D13S317, D5S818 and D21S11) for human cell line authentication. These loci collectively provide a genetic profile with a random match probability of 1 in 2.92×109 and exceed standard ASN-0002 issued by the American Tissue Culture Collection Standards Development Organization Workgroup for cell line authentication.

We have recently introduced a human cell line authentication service using the Promega GenePrint® 10 kit. Data analysis is provided by comparing the loci allele sizes of the test sample with those available at the ATCC database of human cell line STR profiles.

For cell lines not included in the database

  • Users can compare the current profile of a cell line derived from a donor with the profile of the DNA from the same donor.
  • Compare with the profile of an early passage frozen cells.
  • Compare with the profile of the same cell line from different laboratories and, so on.

Here are two useful links from the International Cell Line Authentication Committee (ICLAC)

We also offer mycoplasma detection service at the Cell Center Service lab

(https://www.med.upenn.edu/genetics/cellctr/services/cell_culture_services.shtml).

The assay is based on bioluminescent detection of mycoplasma specific enzymes using MycoAlert kit from Cambrex.

A typical STR profile of a cell line using GenePrint® 10 kit

str profile

Here is a typical output (% match) after searching the ATCC database

80-100% match: test sample related/identical to certain cell line(s) - desired result;

0-55% match: unrelated;

56-79% match: may be contaminated/misidentified, may be related to some known variable cell lines. Needs additional testing with more loci or a different test method.

 output