Abstract
Animal cell lines have become very popular substrates for the production of vaccines and biopharmaceuticals. Characterization of candidate production cell lines is central to ensure product safety and maintenance of consistency in the manufacture of biologicals. Nested PCR and isoenzyme analysis have been used widely to prove the identity and purity of various cell lines and primary cells individually and also after deliberate cross-contamination. The nested PCR based on the Cytochrome b (Cyt b) gene of mitochondrial DNA (Mt DNA) was found to be more sensitive than isoenzyme analysis in detecting low levels of contaminants (as low as 1%). Interestingly, competition between different co-cultured cell lines has shown in one case that cross-contamination need not always results in a mixed cell population. The nested PCR technique for the Cyt b gene described in this study appears to be a potential replacement for isoenzyme analysis and here we demonstrate the PCR method used is sensitive and reliable for cell line authentication in a simple, rapid and reliable format to help assure the authenticity of cell substrates for the production of safe vaccines and biopharmaceuticals.
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Coecke S, Balls M, Bowe G, Davis J, Gstraunthaler G, Hartung T, Hay R, Merten OW, Price A, Schechtman L, Stacey G, Stokes W (2005) Guidance on good cell culture practice. A report of the second ECVAM task force on good cell culture practice. Altern Lab Anim 33:261–287
Dirks WG, MacLeod RAF, Jaeger K, Milch H, Drexler HG (1999) First searchable data base for DNA profile of human cell lines; sequential use of fingerprint techniques for authentication. Cell Mol Biol 45:841–853
Forsell MNE, Li Y, Sundback M, Svehla K, Liljestorm P, Masera JR, Wyatt R, Hedestam GBK (2005) Biochemical and immunogenic characterization of soluble human immunodeficiency virus type 1 envelope glycoprotein trimers expressed by Semliki Forest virus. J Virol 79:10902–10914
Freshney RI (2002) Cell line provenance. Cytotechnology 39:55–67. doi:10.1023/A;1022949730029
Freshney RI (2006) Culture of cells for tissue engineering. Wiley, Hoboken
Gartler SM (1967) Genetic markers as tracers in cell culture. Natl Cancer Inst Monogr 26:167–195
Halton DM, Peterson WD Jr, Hukku B (1983) Cell culture quality control by rapid isoenzymatic characterization. In Vitro 19:16–24
Hay RJ (2000) Animal cell culture, a practical approach. IRL Press, Oxford
Hay RJ (2002) Methods of tissue engineering. Academic Press, New York
Jeffreys AJ, Wilson V, Thein SL (1985) Individual-specific ‘fingerprints’ of human DNA. Nature 316:76–79. doi:10.1038/316076a0
Keith WN (2000) Animal cell culture, a practical approach. IRL Press, Oxford
Liu MY, Lin SC, Liu H, Candal F, Vafai A (2003) Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing. In Vitro Cell Dev Biol Anim 39:424–427
Lopez-Andreo M, Lugo L, Garrido–Pertierra A, Prieto MI, Puyet A (2005) Identification and quantitation of species in complex DNA mixtures by real time polymerase chain reaction. Anal Biochem 339:73–82. doi:10.1016/j.ab.2004.11.045
Losi GC, Ferrari S, Sossi E, Villa R, Ferrari M (2008) An alternative method to isoenzyme profile for cell line identification and interspecies cross-contaminations: cytochrome b PCR-RFLP analysis. In Vitro Cell Dev Biol Animal 44:321–329. doi:10.1007/S11626-008-9125-X
Markovic O, Markovic N (1998) Cell cross-contamination in cell cultures: the silent and neglected danger. In Vitro Cell Dev Biol Anim 34:1–8. doi:10.1007/S11626-998-0040-Y
Masters JR (2000) Human cancer cell lines: facts and fantasy. Nat Rev Mol Cell Biol 1:233–236
Miller OJ, Miller DA, Allderdice PW, Dev VG, Grewal MS (1971) Quinacrine fluorescent karyotypes of human diploid and heteroploid cell lines. Cytogenetics 10:338–346. doi:10.1007/BF02633971
Nelson-Rees W, Flandermeyer RR (1977) Inter-and intraspecies contamination of human breast tumor cell lines HBC and BrCa5 and other cell cultures. Science 195:1343–1344
Nelson-Rees WA, Flandermeyer RR, Hawthorne PK (1974) Banded marker chromosome as indicators of intraspecies cellular contamination. Science 184:1093–1096. doi:10.1126/Science.184.4141.1093
Nelson-Rees WA, Daniels DW, Flandermeyer RR (1989) Contamination of cells in culture. Science 212:446–452
Nims RW, Herbstritt CJ (2005) Cell line authentication using isoenzyme analysis. BioPharm Int 1:1–5
Nims RW, Shoemaker AP, Bauernschub MA, Rec LJ, Harbell JW (1998) Sensitivity of isoenzyme analysis for the detection of interspecies cell line cross-contamination. In Vitro Cell Dev Biol Anim 34:35–39
O’Brien SJ, Shannon JE, Gail MH (1980) A molecular approach to the identification and individualization of human and animal cells in culture: isozyme and allozyme genetic signatures. In Vitro 16:119–135. doi:10.1007/BF02831503
O’Toole CM, Pavey S, Hepburn P, Frank LM (1983) Identity of some human bladder cancer lines. Nature 201:429–430
Ono K, Satoh M, Yoshida T, Ozawa Y, Kohara A, Takeuchi M (2007) Species identification of animal cells by nested PCR targeted to mitochondrial DNA. In Vitro Cell Dev Biol Anim. doi:10.1007/S11626-007-9033-5
Parodi B, Aresu O, Bini D, Lorenzini R, Schena F, Visconti P et al (2002) Species identification and confirmation of human and animal cell lines: a PCR-based method. Biotechniques 32:432–440
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Stacey GN, Bolton BJ, Doyle A (1992) DNA fingerprinting transforms the art of cell authentication. Nature 357:261–262. doi:10.1038/35726/a0
Stacey GN, Hoelzl H, Stephenson JR, Doyle A (1997) Authentication of animal cell cultures by direct visualization of repetitive DNA, aldolase gene PCR and isoenzyme analysis. Biologicals 25:75–85. doi:10.1006/biol.1996.0062
Stacey GN, Masters JR, Hay RJ, Drexler HG, MacLeod RAF, Freshney RI (2000) Cell contamination leads to inaccurate data: we must take action now. Nature 403:356
Steube KG, Grunicke D, Drexler HG (1995) Isoenzyme analysis as a rapid method for the examination of the species identity of cell cultures. In Vitro Cell Dev Biol Anim 31:115–119. doi:10.1007/BF02633971
Stulberg CS, Peterson WR Jr, Simpson WF (1976) Identification of cells in culture. Am J Hematol 1:237–242
Acknowledgments
Our thanks are due to Dr. Glyn N. Stacey for his valuable suggestions, constant support and critical reading of the manuscript. We also thank Dr. Rajan Sriraman and Dr. Mohanasubramanian of Plant Biotechnology and Dr. S. B. Nagendrakumar and Dr. M. Madhanmohan of Foot- and- mouth disease virus laboratory at R and D center, Indian Immunologicals Limited, Hyderabad for their excellent technical support and assistance.
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Ramya, R., Nagarajan, T., Sivakumar, V. et al. Identification of cross-contaminated animal cells by PCR and isoenzyme analysis. Cytotechnology 61, 81–92 (2009). https://doi.org/10.1007/s10616-009-9245-5
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DOI: https://doi.org/10.1007/s10616-009-9245-5