Species identification in cell culture: a two-pronged molecular approach Jason K. Cooper & Greg Sykes & Steve King & Karin Cottrill & Natalia V. Ivanova & Robert Hanner & Pranvera Ikonomi Received: 30 August 2007 / Accepted: 10 September 2007 / Published online: 13 October 2007 / Editor: J. Denry Sato # The Society for In Vitro Biology 2007 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 cumber- some and the data interpretation is often difficult. Further- more, 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 identifica- tion 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 accu- rately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts. Keywords DNA barcode . Sequencing . PCR . Cytochrome C oxidase I (COI) . Species-specific primers Introduction Researchers rely on cell lines as model systems for basic research, standards, and controls. A significant portion of this research is however misleading because cell lines are of a different origin from the one being claimed (Stacey 2000). Several reports have demonstrated evidence of interspecies and intraspecies contamination (Povey et al. 1976; Nelson- Rees et al. 1981). Most notably, cross-contamination and subsequent overgrowth of HeLa cells invalidated many cell culture-based experiments (Nelson-Rees et al. 1981; Masters 2002). In another example, a series of early reports on the establishment and characterization of Hodgkin’ s disease in human cell cultures was marred by misidentifi- cation; three cell lines proved to be nonhuman and, in fact, were derived from owl monkey (Harris et al. 1981). There- fore, as the awareness of the contamination or misidentifi- cation of cell lines increases (Chatterjee 2007), researchers have been implored to ensure the identity and the purity of their cell lines (Langdon 2004; Lincoln and Gabridge 1998; Markovic and Markovic 1998). Emerging genetic methodology offers the most promise in species verification because these methods are easy to use and cost-effective. Analysis of short tandem repeats (STR), although limited to a small number of species origins, has become a valuable tool to establish the identity of cell lines. For interspecies identification of cell cultures, several PCR-based methods have recently been described (Parodi et al. 2002; Liu et al. 2003; Steube et al. 2003). However, the biochemical analysis of isoenzyme polymor- phism, developed over 35 years ago, remains the most common test used, if any testing is performed at all. In Vitro Cell.Dev.Biol.—Animal (2007) 43:344–351 DOI 10.1007/s11626-007-9060-2 J. K. Cooper : G. Sykes : S. King : K. Cottrill : P. Ikonomi (*) ATCC (American Type Culture Collection), 10801 University Boulevard, Manassas, VA 20110, USA e-mail: pikonomi@atcc.org N. V. Ivanova : R. Hanner Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada, N1G 2W1