Results of an online survey conducted by the Global Biological Standards Institute (GBSI), confirmed what many cell scientists already know.

More than half of respondents—52%—“never” perform authentication or other species-related quality control tests on the cell lines used in their experiments. According to survey results, seventy four percent never conduct STR (short-tandem repeat) DNA profiling, the accepted standard for authentication.

Although respondents were more likely to perform some sterility-related microbial contamination quality controls, particularly visual inspections, such measures will not typically detect mycoplasma bacteria-infected cells.

Cell and tissue culture contamination with microbial contamination and other cell types not only affects the culture in question, but also compromises overall reproducibility of experimental results.

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With the continuing increase in the use of cell culture for biological research, vaccine production, and production of therapeutic proteins for personalized medicine and emerging regenerative medicine applications, culture contamination remains a significant concern. Based on submissions to major cell repositories in the last decade, it is estimated that between 18% and 36% of cell lines may be contaminated or misidentified.

In 2008 for example, 40 human thyroid cancer cell lines were analyzed by STR profiling. Only 23 unique profiles were obtained, and many of the cross-contaminating cell lines were not thyroid in origin. These cell lines had been previously used for two decades in thyroid cancer research.

Due to the scope of this problem, the NIH and many journals now recommend or require cell-line authentication prior to grant approval or acceptance for publication, making it critical for scientist using cell cultures for research.

Avoiding contamination with other cell types

General considerations

  • Maintain good cell culture practices in general: to help ensure maintenance of a single cell type handle only a single cell line in a bio-safety hood at any one time.
  • Clean the workspace thoroughly between using it for specific cell lines.
  • Dedicate equipment to a single cell line including bottles and aliquots of cell culture medium and other reagents.
  • Clearly label bottles; aliquots should also be clearly labeled and never shared between cell lines.
  • Empty waste receptacles regularly and use a disinfectant effective against both microbes and cell cultures.

Taking specific precautions

  • Obtain cell lines from reputable cell banks.
  • Develop and maintain a controlled cell banking regime of master and working cell banks. Adherence to these routines helps avoid contamination, cross-contamination, and genetic and phenotypic instability that can increase with each successive subculture.
  • Perform authentication tests including STR profiling, isoenzyme analysis, and contamination tests.
  • Avoid cell line transfers to colleagues. If transfers do occur, cells should be accompanied with comprehensive documentation verifying the integrity of the material.

Obtaining cell lines from reputable cell banks, periodically checking the characteristics of the cell lines, and practicing good aseptic technique will help avoid cross-contamination. DNA fingerprinting, karyotype analysis, and isotype analysis can confirm the presence or absence of cross-contamination in your cell cultures.

Continuous monitoring of cultured cells

Regular checks of cell-line morphology and growth characteristics by phase contrast microscopy and comparison with reference images can indicate early problems. Such clues include Identification of unexpected morphology or growth characteristics in a cell line soon after recovery from frozen storage that could mean recovery of the wrong vial from storage. Printed labels including the cell line name in its correct format must have adhesive suitable for low temperature storage, as inappropriately adhered labels will detach, leaving an unlabeled vial in the inventory.

The American Type Culture Collection (ATCC) has developed a Standard for human cell-line authentication that recommends STR profiling. STR analysis can be performed in most laboratories that can execute molecular techniques providing an easy, low cost, and reliable method for the authentication of human cell lines.

A combination of regular identity testing and vigilance of good cell culture practices is required to provide valid cell cultures and to ensure that research is acceptable for peer review. Failure to adhere to these guidelines will lead to an undesirable legacy and invalid research.

References

1. Passim, et al. The costs of using unauthenticated, over-passaged cell lines: how much more data do we need? Biotechniques. 2007 Nov;43(5):575, 577-8, 581-2 .

2. Edward Burnett, Ph.D.; James Cooper, Detection and Prevention of Cell Line Cross-Contamination: Biowire Spring 2012, 19–2.1

3. Shweppe et al: Deoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification. The Journal of Clinical Endocrinology & Metabolism, Volume 93, Issue 11, 1 November 2008, pages 4331–4341.

4. Masters, JR et al. Short tandem repeat profiling provides an international reference standard for human cell lines. Proc Natl Acid Sci U S A. 2001 Jul 3; 98(14): 8012–8017.

5. Freedman, LP et al.Changing the Culture of Cell Culture: Applying Best Practices and Authentication to Ensure Scientific Reproducibility.Oct 01, 2015. Bio Pharm International Volume 28, Issue 10, pages 14–21.