Abstract

Tissue culture cell lines are a critical reagent for infectious disease research and comparative biomedical research, and they can serve as a repository of genetic information for wildlife conservation. While a wide variety of cell lines are available from humans, domesticated and laboratory mammals, and even fish, cell lines from wildlife species—and particularly reptiles and amphibians—are extremely limited. As part of an ongoing project to increase the availability and access to reagents for wildlife disease research, we are establishing continuously dividing cell lines from reptile and amphibian species. Cell lines are derived from primary tissues collected at the time of necropsy, portions of surgical biopsies, or from embryonic tissues culled for management purposes. Primary cell growth was routinely observed from a variety of tissues, including heart, spleen, kidney, skeletal muscle, liver, and gonad. Continued selection for stable, dividing cells of homogenous morphology was achieved through passage in growth conditions determined optimal for the class of the origin host. Aliquots of cell lines were routinely archived in liquid nitrogen to create low passage stocks. After 10–12 serial passages characterized by stable, predictable growth of cells representing a single cellular morphology, the cell line was considered established, and aliquots of working stock cells were archived in liquid nitrogen. Final cell line characterization includes confirmation of host origin by sequencing of the cytochrome oxidase 1 gene, and immunohistochemical characterization to determine histogenesis when possible. At the time of submission, cell lines have been successfully established for various tissues from a variety of reptile species, including snakes (n=6), turtles/tortoises (n=4), crocodilians (n=4), and lizards (n=2). Additional cell lines from other species of reptiles and amphibians are currently in various stages of development. Cell lines will be cataloged in a publicly accessible virtual database to permit and promote the dissemination of these reagents to improve disease diagnostics and characterization of captive and free-ranging wildlife species.

Acknowledgments

The authors thank John S.L. Parker, James F.X. Wellehan, Edward Dubovi, Amy Alexander, Darryl Heard, Jane Christman, Ricardo de Matos, Noha Abou-Madi, Danielle Tarbert, the Zoologic Medicine Service at Cornell University, the Zoologic Medicine Service at the University of Florida, Penny Felski, Kurt Volle, Jess Lovstad, Kathryn Gamble, Ben Parrott, the Morris Animal Foundation, and the Atkinson Center at Cornell University.