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Cell Line Authentication – Why it Matters

As a biological resource center, ATCC and other recognized cell banks comprehensively perform authentication and quality-control tests on all distribution lots of cell lines.

Authentication of a cell line is the sum of the process by which a line’s identity is verified and shown to be free of contamination from other cell lines and microbes. Using standardized techniques, authentication enables communication among all users about the resource and ensures valid, reproducible experimental results. Without periodic testing, over-subcultured, misidentified or cross-contaminated cell lines are released into the research arena resulting in spurious data.1 Clearly, the research community is better served by the use of tested material. However, guidelines for general testing of cell lines currently do not exist—especially for lines not obtained from a cell bank or are derived locally. In this article, ATCC seeks to recommend basic benchmark verification tests that can be employed by any lab and included in publication. ATCC seeks to engage the research community in an ongoing dialogue about this topic, including suggestions and ideas about the tests set forth herein.

Obtaining and using low-passage cell lines from ATCC is a sure way to work and publish with confidence. At the time of publishing with an ATCC obtained cell line, the designation, the ATCC catalog number and the passage numbers under which experiments were conducted should be included in the materials and methods (e.g., NIH/3T3, ATCC® No. CRL-1658™, passage number XX – YY).

ATCC recognizes that many cell lines used in basic and biomedical research are not available at ATCC or another cell bank. In some cases, these lines have undergone little or no authentication testing. In other cases, only outdated testing has been conducted. When performing research with these cell lines, it is good cell culture practice to conduct fundamental tests to ensure that the lines are uncontaminated and correctly identified. When research with this material is published, details of the tests can and should be submitted to the journal editor and included in the materials and methods section of a manuscript.

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Recommended Tests

Morphology check by microscope — Cellular morphology refers to the optical observation of a magnified cell culture. This can be the simplest and most direct method used to identify the state of cells. Obtaining morphology information from comparative observations both at high and low culture densities depends on knowledge of several factors. Morphology can vary between lines depending on the health of the cells and, in some cases, the differentiation state. Morphology can change with plating density as well as with different media and sera combinations. Cell morphology is best monitored through frequent, brief observations. In general, if a culture has an unusual appearance, there is likely a problem. It is recommended that researchers be alert during periodic morphology checks and maintain cell morphology images for comparisons.

CCL-1 HTB-38HB-9596

Three different ATCC cell lines at high densities.

Growth curve analysis —Evaluation of cell proliferation can yield valuable information about a culture’s response to a stimulus. Variable growth or sudden decreases or increases are a sign that something may be amiss with your cell lines. Establishing baselines and quantifying cell culture growth is a crucial element for monitoring the consistency of the culture and determining a number of other factors, such as the best time to subculture, the optimum dilution, and the estimated plating efficiency at various cell densities. Growth curve analysis can also help determine population doubling times and should be performed routinely when enzymatic or functional analysis is imminent. Using cell lines with consistent growth properties should be pursued as a rule.

Species verification by Isoenzymology — Isoenzyme analysis is used to verify the species of origin. Isoenzyme specimens are differentiated based on electrophoretic properties. Distribution patterns of a group of enzymes are characteristic of a particular species which simultaneously confirms the species identity and reveals contamination by another line of different species. Isoenzyme testing is available in a kit format (Authentikit™ system) from the company Innovative Chemistry (www.authentikit.com) and a detailed protocol may also be found in Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, 5th ed. Pp. 275 – 278 (ATCC® No. 30-3001).

Identity verification with STR analysis (DNA fingerprinting) for human cell lines — DNA fingerprinting is a powerful tool in determining the identity and uniqueness of a human line. Short tandem repeat (STR) profiling establishes a DNA fingerprint for every human cell line and may be used as a record of the line. ATCC STR profiling uses multiplex PCR to simultaneously amplify the amelogenin gene and eight of the most informative polymorphic markers in the human genome. The pattern of repeats results in a unique STR identity profile for each cell line analyzed. The profile can be used as a baseline for comparison with future tests. ATCC uses the Promega PowerPlex® 1.2 system and the Applied Biosystems Genotyper 2.0 software for analysis of the amplicons.

Identity verification with STR analysis (DNA fingerprinting) for Mouse cell lines —While human cell line authentication has been addressed with a short tandem repeat (STR) profiling, up until now there has not been a validated method for identifying mouse cell lines beyond the species level. ATCC now provides a powerful STR profiling service that authenticates mouse cell lines and detects human and African green monkey cell contamination. Like the human STR profiling service, we use the Promega PowerPlex® 1.2 system and the Applied Biosystems Genotyper 2.0 software for analysis of the amplicons.

Mycoplasma detection — A major problem in cell culture, mycoplasma infection can have adverse effects on cell lines; altering cell behavior and metabolism in many ways.2-7 Periodic assays to detect mycoplasma are critical for all continuous cell lines. A relatively easy and reliable biochemical method for detecting mycoplasma in a cell culture is to use Hoechst 33258, a fluorescent dye that binds specifically to DNA. Fluorescent Hoechst staining reveals mycoplasma infections through their characteristic patterns of extracellular particulate or filamentous fluorescence at 500X magnification.

Conclusion

While tests are always a good idea, your intuitions should not be ignored. When in doubt about the state of your cell lines, start with a new vial from your cell bank. By treating your cell lines as standard research components and providing verification benchmarks in publication, the research community is better served and information exchange between colleagues is enhanced. The result will be more reliable and reproducible experimental data.

NOTE: When publishing with ATCC acquired cell lines, always reference the line with the common name followed by the ATCC catalog number; e.g., NIH/3T3, ATCC® No. CRL-1658™.

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References
  1. Chatterjee R. Cases of Mistaken Identity. Science. 315:928-931, 2007. PMID: 17303729
  2. Drexler HG et al. Mix-ups and mycoplasma: the enemies within. Leukemia Research. 26(4): 329 333, 2002. PMID: 11839374
  3. Kagemann G et al. Impact of Mycoplasma hyorhinus infection of L-arginine metabolism: differential regulation of the human and murine iNOS gene. Biological Chemistry. 386 (10): 1055- 1063, 2005. PMID: 16218877
  4. Lincoln CK et al. Cell culture contamination: sources, consequences, prevention and elimination. Methods in Cell Biology. 57: 49-65, 1998. PMID: 9648099
  5. McGarrity GJ et al. Cell culture mycoplasmas. In: The Mycoplasma, Vol. IV. Razin S and Barile MF, eds. New York: Academic Press. 353-390, 1985.
  6. Mirjalili A et al. Microbial contamination of cell cultures: a 2 year study. Biologicals. 33(2): 81-85, 2005. PMID: 15939285
  7. No authors listed. Contamination of cell lines—a conspiracy of silence. Lancet Oncology. 2(7): 393, 2001. PMID: 11905730
  8. Esquenet M et al. LNCaP prostatic adenocarcinoma cells derived from low and high passage numbers display divergent responses not only to androgens but also to retinoids. J. Steroid Biochem. Mol. Biol. 62(5-6): 391-399, 1997. PMID: 9449242
  9. Briske-Anderson MJ et al. Influence of culture time and passage number on the morphological and physiological development of Caco-2 cells. Proc. Soc. Exp. Biol. Med. 214(3): 248-257, 1997. PMID: 9083258
  10. Yu H et al. Evidence for diminished functional expression of intestinal transporters in Caco-2 cell monolayers at high passages. Pharm. Res. 14(6): 757-762, 1997. PMID: 9210193
  11. Sambuy Y et al. The Caco-2 cell line as a model of the intestinal barrier: Influence of cell- and culture-related factors on Caco-2 functional characteristics. Cell Biol. Toxicol. 21(1): 1-26, 2005. PMID: 15868485
  12. Wenger SL et al. Comparison of establishe d cell lines at different passages by karyotype and comparative genomic hybridization. Biosci. Rep. 24(6): 631-639, 2004. PMID: 16158200
  13. MacLeod RA et al. Identity of original and late passage Dami megakaryocytes with HEL erythroleukemia cells shown by combined cytogenetics and DNA fingerprinting. Leukemia. 11 (12): 2032-2038, 1997. PMID: 9447816