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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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.
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
- DNA barcode
- Cytochrome C oxidase I (COI)
- Species-specific primers