Cell Line Authentication and Characterisation

Abstract

Cell lines are widely used in biomedical research as surrogates for human tissues and for physiological studies on live cells. Despite being easy to maintain and manipulate, cell lines must be carefully characterised. When a new cell line is acquired, and when preparing a new cell bank for storage, it is mandatory to confirm the identity of the cells and ensure that they are free of Mycoplasma and other microbial contaminants. These tests must be repeated whenever another cell bank is prepared. Cell lines must be replaced from the bank within 10 passages or 3 months of continuous growth, whichever is the shorter period. Moreover, it is important that cells are examined microscopically for morphological changes and contamination every time they are taken out of the incubator.

Key Concepts:

  • Every time cells are taken out of the incubator, they must be examined microscopically for changes in morphology or the presence of microbial and cellular contamination.

  • Every time a new cell line is acquired or a cell stock is frozen for storage, it should be authenticated by interspecies (DNA barcoding) and intraspecies (STR profiling) identification.

  • Every time a new cell line is acquired or a cell stock is cryopreserved, it is essential to check for Mycoplasma contamination (and other microbial contamination if tests are available).

  • An established cell line should never be grown continuously for more than 3 months or 10 passages, whichever is the shorter period.

  • All cell stocks entering the tissue culture laboratory must be kept in quarantine until microbial and cellular contaminations are excluded.

  • If there are any doubts concerning the authenticity of cells, consult the ICLAC (International Cell Line Authentication Committee) database of misidentified cell lines – http://iclac.org/databases/cross‐contaminations/

  • When obtaining cells from a cell bank or another reliable source, always ask for proof of authenticity and lack of Mycoplasma contamination.

  • Always follow good tissue culture practices.

  • Determine key characteristics and morphology at various magnifications and densities.

  • STR profiling (human) and DNA barcoding (animal) are commercially available, and are cheap, robust and reliable methods of authenticating cell lines.

Keywords: STR profiling; DNA barcoding; cell line cross‐contamination; Mycoplasma detection; cell culture

Figure 1.

COI analysis (DNA barcoding) revealing the interspecies identity of 14 common animal species cell lines. Each species is characterised by a specific amplicon size, which is used for identification. Amplicons generated from PCR amplification of the COI gene of cell lines representing 14 different species were separated by agarose gel electrophoresis and stained with ethidium bromide.

Figure 2.

Electropherograms of STR DNA profiles of two unrelated cell lines: (a) K562 (chronic myelogenous leukaemia) and (b) WS1 (skin fibroblast). Cells spotted onto an FTA® card were amplified by multiplex PCR, separated by capillary electrophoresis, and analysed by Genotyper® software. The peaks at each locus were converted to numerical values based on allelic ladders. The numerical values or the unique STR profile of each cell line can be used to establish a database.

Figure 3.

Indirect Hoechst staining of Vero cells. Cell suspensions from actively growing cells were incubated with Vero indicator cells and stained with Hoechst stain. (a) Vero cells positive for Mycoplasma contamination. (b) Vero cells negative for Mycoplasma contamination. In both panels, the Vero cells nuclei are visible. In panel (a), contamination is visualised by extracellular fluorescence emission from the Mycoplasma attached to the cell surface (arrow).

Figure 4.

Photomicrograph of amplicons separated by agarose gel electrophoresis and stained with ethidium bromide. Mycoplasma strains can be speciated by fragment size. Lanes 1 and 10: 100 bp DNA ladder, lane 2: Acholeplasma laidlawii, lane 3: Melongena pirum, lane 4: Mycoplasma hyorhinis, lane 5: Mycoplasma salivarium, lane 6: Mycoplasma arginini, lane 7: Mycoplasma hominis, lane 8: Mycoplasma orale, lane 9: Mycoplasma fermentans.

close

References

Almeida JL, Hill CR and Cole KD (2014) Mouse cell line authentication. Cytotechnology 66: 133–147.

Barallon R, Bauer SR, Butler J et al. (2010) Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells and tissues. In Vitro Cellular & Developmental Biology – Animal 46(9): 727–732.

Bel‐Price A and Coecke S (2011) Guidance on good tissue culture practice (GCCP). Neuromethods 56: 1–25.

Capes‐Davis A, Reid YA, Kline MC et al. (2013) Match criteria for human cell line authentication: where do we draw the line? International Journal of Cancer 132(11): 2510–2519.

Castro F, Dirks WG, Fahnrich S et al. (2013) High‐throughput SNP‐based authentication of human cell lines. International Journal of Cancer 132(2): 308–314.

CBER (2009) Guidance for Industry: Current Good Tissue Practice (CGTP) and Additional Requirements for Manufacturers of Human Cells, Tissues, and Cellular and Tissue‐Based Products (HCT/Ps). U.S. Department of Health and Human Services, FDA.

Chen J, Pennamaneni D, Komaragiri S, Wright C and Hawthorne B (2011) Selecting cell lines for SNP human identification assay development. Atlas Journal of Biotechnology 1(1): 21–26.

Cooper JK, Sykes G, King S et al. (2007) Species identification in cell culture: a two‐pronged molecular approach. In Vitro Cellular & Developmental Biology –Animal 43(10): 344–351.

Demichelis F, Greulich H, Macoska JA et al. (2008) SNP panel identification assay (SPIA): a genetic‐based assay for the identification of cell lines. Nucleic Acids Research 36(7): 2446–2456.

Freshney RI (2011) Culture of Animal Cells: A Manual of Basic Techniques and Specialized Applications, 6th edn. Hoboken, NJ: John Wiley & Sons.

Gartler SM (1968) Apparent Hela cell contamination of human heteroploid cell lines. Nature 24(5130): 750–751.

Gilbert DA, Reid YA, Gail MH et al. (1990) Application of DNA fingerprints for cell‐line individualization. American Journal of Human Genetics 47(3): 499–514.

Hay RJ (1992) Testing for microbial contaminants – bacteria and fungi. In: Hay RJ, Caputo J and Macy ML (eds) ATCC Quality Control Methods for Cell Lines, 2nd edn, pp. 19–33. Rockville, MD: American Type Culture Collection.

Hay RJ (1998) Cell banking and authentication. In: Brown F, Griffiths E, Horaud JC and Petricciani JC (eds) Safety of Biological Products Prepared from Mammalian Cell Culture, Developments in Biological Standardization, vol. 93, pp. 15–19. Basel: Karger.

Jeffreys AJ, Wilson V and Thein SL (1985) Hypervariable ‘minisatellite’ region in human DNA. Nature 314: 67–73.

Masters JR, Thompson JA, Daly‐Burns B et al. (2001) Short tandem repeat profiling provides an international reference standard for human cell lines. Proceedings of the National Academy of Sciences of the USA 98(14): 8012–8017.

McGarrity GJ, Sarama J and Vanaman V (1985) Cell culture techniques. ASM News 51: 170–183.

McLaren RS, Reid Y and Storts DR (2013) Human cell line authentication: the critical first step in any project using human cell lines. Methods in Molecular Biology 963: 341–353.

Reid Y, Storts D, Riss T and Minor L (2013) Authentication of human cell lines by STR DNA profiling analysis. In: Sittampalam GS, Gal‐Edd N, Arkin M et al. (eds) Assay Guidance Manual, pp. 1–22. Bethesda, MD: Eli Lilly & Company and the National Center for Advancing Translational Sciences. (http://www.ncbi.nlm.nih.gov/books/NBK144066/)

Rottem S and Barile MF (1993) Beware of Mycoplasmas. Trends in Biotechnology 11: 143–150.

Young L, Sung J, Stacey G and Masters JR (2010) Detection of Mycoplasma in cell cultures. Nature Protocols 5(5): 929–934.

Further Reading

Chen TR (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Experimental Cell Research 104: 255–262.

Lincoln CK and Gabridge MA (1998) Cell culture contamination: sources, consequences, prevention and elimination. In: Mather JP and Barnes D (eds) Animal Cell Culture Methods, pp. 50–64. New York, NY: Academic Press.

McGarrity GJ (1976) Spread and control of mycoplasmal infection of cell cultures. In Vitro 12: 643–648.

Nelson‐Rees WA, Flandermeyer RR and Hawthorne PK (1975) Distinctive banded marker chromosomes of human tumor cell lines. International Journal of Cancer 16: 74–82.

Peterson WD, Simpson WF and Hukku B (1979) Cell culture characterization: monitoring for cell identification. Methods in Enzymology 58: 164–178.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Masters, John R, and Reid, Yvonne A(Oct 2014) Cell Line Authentication and Characterisation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002559.pub2]