Authenticating cell lines in ophthalmic research laboratories.

Authentication of cell lines in biomedical research has been elevated to a very high priority. In a review of the literature, Lacroix examined the issue of cross-contamination of cell lines including the well-known contamination of cell lines with HeLa cells, and the misidentification of the ECV304 cell line as “immortalized endothelial cells” when these cells in fact originated from T24 bladder carcinoma cells. Lacroix estimated that between 18% and 36% of cell lines have been misclassified. One survey at a large research institution suggested that fewer than 50% of researchers authenticate their cell lines. Nardone proposed recently that identification of cell lines be required of investigators before grants are awarded, and the National Institutes of Health subsequently called for researchers to authenticate cell lines as a prerequisite for grant funding. Cell lines are widely used in ophthalmic investigations. The authors’ own research efforts have been focused on the biology of metastasis of uveal melanoma, and in the field of ocular oncology research, the use of cell lines is critical for at least two reasons. First, there are no animal models of spontaneous uveal melanoma that faithfully replicate the behavior of the human disease. Transgenic models of uveal melanoma do not reproduce histogenesis of human uveal melanoma, and spontaneous uveal melanomas develop too sporadically in nature to be valuable in research, or the animal melanoma tumors vary significantly from their human counterparts. Therefore, uveal melanoma researchers have increasingly relied on the implantation of animal and human melanoma cell lines into animals to model the behavior of human uveal melanoma to improve imaging techniques and to investigate immunologic and molecular mechanisms of tumor behavior. Second, mindful of the reality that statistical associations between histologic characteristics and outcome do not always indicate causality, the availability of cell lines allows ophthalmic researchers to advance from correlative studies to mechanistic investigations using advanced molecular methodologies. For example, All-Ericsson et al. identified the association between the expression of insulin-like growth factor-1 by immunohistochemistry in tissue sections of uveal melanoma and adverse outcome and also demonstrated decreased tumor cell viability in uveal melanoma cell lines after interfering with the expression of ILGRF-1, pointing to the molecular significance of this histologic finding. Later, Girnita et al., from the same group, expanded on this finding to suggest a novel therapeutic approach to uveal melanoma using the pharmacological manipulation of ILGRF-1 in human uveal melanoma cell lines. It is possible that the behavior of long-term cell lines is not representative of the spectrum of phenotypic behavior of neoplasms, because those lines that are established in vitro have been selected for this artificial growth environment. In addition, the phenotypic behavior of cancer cell lines can change over time. And yet, despite these limitations, there are few satisfactory alternatives to the use of long-term cell lines in modeling uveal melanoma behavior in animals or testing molecular approaches to the modifying of tumor cell behavior. Some investigators have preferred to work with short-term primary cultures to circumvent these limitations of long-term cell lines, but short-term cultures cannot be reliably shared between investigative groups, making it difficult to obtain independent validation of results on common biological substrates. Therefore, investigators have developed mechanisms for sharing long-term cell lines between groups. In many areas of biomedical research, investigators obtain long-term cell lines from centralized repositories. However, many cell lines developed for ophthalmic research are not available from these agencies. For example, uveal melanoma cell lines are not identified in the inventories of the American Tissue Culture Collection (ATCC, Manassas, VA, www.atcc. org), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany; www.dsmz.de), the European Collection of Cell Cultures (ECACC; www.ecacc. org.uk), or the Japanese Collection of Research Bioresources (JCRB; Osaka, Japan; http://cellbank.nibio.go.jp). The European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI; Cambridge, UK) Web site (http://www. ebi.ac.uk/ipd/estdab/directory.html) lists only four uveal melanoma cell lines: OCM3, OCM8, 92.1, and Mel-202. To complicate matters, it is relatively difficult to establish new long-term uveal melanoma cell lines. One investigator noted that more than 60% of attempts to establish such lines were unsuccessful. Therefore, as in the case of many ophthalmic research endeavors, ophthalmic oncology researchers have shared uveal melanoma cell lines with each other. The relatively few uveal melanoma cell lines available for research have been widely circulated, but it is clear from scanning the methods sections of publications in this field that the source of the cell line does not always represent the investigator who actually developed the cell line, indicating the existence of secondary and even tertiary distributions of these scarce but vital resources. The underlying assumption behind this collegial sharing of resources is simple: A named cell line used in the United States is identical with the cell line being used in Europe or Asia with the same name. One also assumes that human lines are indeed human. We are aware of the practice of propagating uveal melanoma cell lines in immunosuppressed mice, introducing the risk that murine cells may contaminate cultures or that murine pathogens can infect culFrom Oakland University William Beaumont School of Medicine, Rochester, Michigan; the Department of Pathology, University of Illinois at Chicago, Chicago, Illinois; the Department of Ophthalmology, Leiden University Medical Centre, Leiden, the Netherlands; and the Department of Ophthalmology, Hadassah-Hebrew University Medical, Jerusalem, Israel. Supported by National Eye Institute Grant R01-EY10457. Submitted for publication May 22, 2008; revised June 25, 2008; accepted September 11, 2008. Disclosure: R. Folberg, None; S.S. Kadkol, None; S. Frenkel, None; K. Valyi-Nagy, None; M.J. Jager, None; J. Pe’er, None; A.J. Maniotis, None Corresponding author: Robert Folberg, Oakland University William Beaumont School of Medicine, 478 O’Dowd Hall, 2200 N. Squirrel Road, Rochester, MI 48309-4401; [email protected].