Characterization of the First Turtle Organoids: A Model for Investigating Unique Adaptations with Biomedical Potential

Painted turtles are remarkable for their well-developed freeze tolerance and associated resilience to hypoxia/anoxia, oxidative stress, and ability to supercool. They are, therefore, are an ideal model for biomedical research on hypoxia-induced injuries (such as strokes), tissue cooling during extensive surgeries, and organ cryopreservation. Yet, the seasonal reproduction and slow maturation of turtles hinder basic and applied biomedical research. To overcome these limitations, we have developed for the first time, adult stem cell-derived turtle hepatic organoids, which provide 3D self-assembled structures that closely mimic their original tissue and allow for in vitro testing and experimentation without constantly harvesting donor tissue and screening offspring. Our pioneering work with turtles represents the first for this vertebrate Order and complements the only other organoid lines from non-avian reptiles, which were produced from snake venom glands. Here we report the isolation and characterization of hepatic organoids derived from painted, snapping, and spiny softshell turtles spanning ~175 million years of evolution, with a subset being preserved in a biobank. Morphological and transcriptomics revealed organoid cells resembling cholangiocytes, which was then compared to the tissue of origin. Deriving organoids from multiple turtle species and life stages demonstrates that our organoid culturing techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently missing in most herpetological research. When combined with genetic editing, this platform will further support studies of genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, among others. We discuss the unique abilities of painted turtles, including their overwintering potential, an adaptation with important implications for ecological, evolutionary, and biomedical research. ### Competing Interest Statement K. Allenspach is a co-founder of LifEngine Animal Health and 3D Health Solutions. She serves as a consultant for Ceva Animal Health, Bioiberica, LifeDiagnostics, Antech Diagnostics, Deerland Probiotics, Christian Hansen Probiotics, Purina, and Mars. J.P. Mochel is a co-founder of LifEngine Animal Health (LEAH) and 3D Health Solutions. Dr. Mochel is a consultant for Ceva Animal Health, Ethos Animal Health, LifEngine Animal Health and Boehringer Ingelheim. C. Zdyrski is the Director of Research and Product Development at 3D Health Solutions. A. Ralston is an employee at 3D Health Solutions. Other authors do not have any conflict of interest to declare.