Abstract
Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the “barcode region” by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.
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Alexander, D.S.; Sipley, J.D.; Quigley, J.P. Autoactivation of avian urokinase-type plasminogen activator (uPA). A novel mode of initiation of the uPA/plasmin cascade. J Biol Chem 273(13):7457–61; 1998.
Buehring, G.C.; Eby, E.A.; Eby M.J. Cell line cross-contamination: how aware are Mammalian cell culturists of the problem and how to monitor it? In Vitro Cell Dev Biol Anim 40(7):211–5; 2004.
Chatterjee, R. Cell biology. Cases of mistaken identity. Science 315(5814):928–31; 2007.
Cywinska, A.; Hunter, F.F.; Hebert, P.D. Identifying Canadian mosquito species through DNA barcodes. Med Vet Entomol 20(4):413–24; 2006.
Farris, A.D.; Koelsch, G.; Pruijn, G.J.; van Venrooij, W.J.; Harley, J.B. Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis. Nucleic Acids Res 27(4):1070–8; 1999.
Folmer, O.; Black, M.; Hoeh, W.; Lutz, R.; Vrijenhoek, R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3(5):294–9; 1994.
Harris, N.L.; Gang, D.L.; Quay, S.C.; Poppema, S; Zamecnik, P.C.; Nelson-Rees, W.A.; O’Brien, S.J. Contamination of Hodgkin’s disease cell cultures. Nature 289(5795):228–30; 1981.
Hay, R.J.; Chen, T.J.; Macy, M.L.; Reid, Y.A. Replay to “Cell, lines and DNA fingerprinting”. In Vitro Cell Dev Biol 28A:593–594; 1992.
Hebert, P.D.; Cywinska, A.; Ball, S.L.; deWaard, J.R. Biological identifications through DNA barcodes. Proc Biol Sci 270(1512):313–321; 2003a
Hebert, P.D.; Ratnasingham, S.; deWaard, J.R. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci 270 Suppl 1:S96–9; 2003b.
Hebert, P.D.; Penton, E.H.; Burns, J.M.; Janzen, D.H.; Hallwachs, W. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci U S A 101(41):14812–7; 2004
Ivanova, N.V.; deWaard, J.R.; Hebert, P.D.N. An inexpensive, automation-friendly protocol for recovering high-quality DNA. Molecular Ecology Notes 6:992–1002; 2006.
Ivanova, N.V.; Zemlak, T.S.; Hanner, R.H.; Hebert, P.D.N. Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes 7(4):544–548; 2007.
Kerr, K.C.R.; Stoeckle, M.Y.; Dove, C.J.; Weight, L.A.; Francis, C.M.; Hebert, P.D.N. Comprehensive DNA barcode coverage of North American birds. Mol Ecol Notes 7(4):535–543; 2007.
Langdon, S.P. Cell culture contamination: an overview. Methods Mol Med 88:309–17; 2004.
Lincoln, C.K.; Gabridge M.G. Cell culture contamination: sources, consequences, prevention, and elimination. Methods Cell Biol 57:49–65; 1998.
Liu, M.Y.; Lin, S.C.; Liu, H.; Candal, F.; Vafai, A. Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing. In Vitro Cell Dev Biol Anim 39(10):424–7; 2003.
Lorenz, J.G.; Jackson, W.E.; Beck, J.C.; Hanner, R. The problems and promise of DNA barcodes for species diagnosis of primate biomaterials. Philos Trans R Soc Lond B Biol Sci 360(1462):1869–77; 2005.
Marcovici, M.; Prier, J.E.. Enhancement of St. Louis arbovirus plaque formation by neutral red. J Virol 2(3):178–81; 1968.
Markovic, O.; Markovic, N. Cell cross-contamination in cell cultures: the silent and neglected danger. In Vitro Cell Dev Biol Anim 34(1):1–8; 1998.
Masters, J.R. HeLa cells 50 years on: the good, the bad and the ugly. Nat Rev Cancer 2(4):315–9; 2002.
Nelson-Rees, W.A.; Daniels, D.W.; Flandermeyer, R.R. Cross-contamination of cells in culture. Science 212(4493):446–52; 1981.
Parodi, B.; Aresu, O.; Bini, D.; Lorenzini, R.; Schena, F.; Visconti, P.; Cesaro, M.; Ferrera, D.; Andreotti V.; Ruzzon T. Species identification and confirmation of human and animal cell lines: a PCR-based method. Biotechniques 32(2):432–4, 436, 438–40; 2002.
Povey, S.; Hopkinson, D.A.; Harris, H.; Franks, L.M. Characterisation of human cell lines and differentiation from HeLa by enzyme typing. Nature 264(5581):60–3; 1976.
Ratnasingham, S.; Hebert, P. The Barcode of Life Database. Mol. Ecol. Notes 7(3):355–364; 2007.
Ryder, O.A.; McLaren, A.; Brenner, S.; Zhang, Y.P.; Benirschke, K. DNA banks for endangered animal species. Science 288(5464):275–7; 2000.
Sick, C.; Schultz, U.; Munster, U.; Meier, J.; Kaspers, B.; Staeheli, P. Promoter structures and differential responses to viral and nonviral inducers of chicken type I interferon genes. J Biol Chem 273(16):9749–54; 1998.
Smith, M.A.; Fisher, B.L.; Hebert, P.D. DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar. Philos Trans R Soc Lond B Biol Sci 360(1462):1825–34; 2005
Stacey, G.N. Cell contamination leads to inaccurate data: we must take action now. Nature 403(6768):356; 2000.
Steube, K.G.; Meyer, C.; Uphoff, C.C.; Drexler, H.G. A simple method using beta-globin polymerase chain reaction for the species identification of animal cell lines—a progress report. In Vitro Cell Dev Biol Anim 39(10):468–75; 2003.
Ward, R.D.; Zemlak, T.S.; Innes, B.H.; Last, P.R.; Hebert, P.D. DNA barcoding Australia’s fish species. Philos Trans R Soc Lond B Biol Sci 360(1462):1847–57; 2005.
Wolf, K.; Burke, C.N.; Quimby, M.C. Duck viral enteritis: microtiter plate isolation and neutralization test using the duck embryo fibroblast cell line. Avian Dis 18(3):427–34; 1974.
Acknowledgments
Research at Guelph was supported through funding to the Canadian Barcode of Life Network from Genome Canada (through the Ontario Genomics Institute), NSERC and other sponsors listed at http://www.BOLNET.ca. The authors thank Jeff Benson for his help with the sequencing analysis.
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Editor: J. Denry Sato
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Cooper, J.K., Sykes, G., King, S. et al. Species identification in cell culture: a two-pronged molecular approach. In Vitro Cell.Dev.Biol.-Animal 43, 344–351 (2007). https://doi.org/10.1007/s11626-007-9060-2
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DOI: https://doi.org/10.1007/s11626-007-9060-2