Identification of cross-contaminated animal cells by PCR and isoenzyme analysis
- 166 Downloads
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.
KeywordsCross-contamination Cyt b gene Isoenzyme analysis Nested PCR
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.
- 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 33:261–287Google Scholar
- 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 45:841–853Google Scholar
- Freshney RI (2006) Culture of cells for tissue engineering. Wiley, HobokenGoogle Scholar
- Gartler SM (1967) Genetic markers as tracers in cell culture. Natl Cancer Inst Monogr 26:167–195Google Scholar
- Hay RJ (2000) Animal cell culture, a practical approach. IRL Press, OxfordGoogle Scholar
- Hay RJ (2002) Methods of tissue engineering. Academic Press, New YorkGoogle Scholar
- Keith WN (2000) Animal cell culture, a practical approach. IRL Press, OxfordGoogle Scholar
- Nims RW, Herbstritt CJ (2005) Cell line authentication using isoenzyme analysis. BioPharm Int 1:1–5Google Scholar
- 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 32:432–440Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar