Cell Line Authentication and Characterisation


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.



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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]