Generation and cryopreservation of feline oviductal organoids

Next-generation in vitro culture model systems are needed to study the reproductive pathologies that affect domestic animals. These 3D culture models more closely mimic normal physiological function to allow a greater understanding of reproductive pathology and to trial therapeutics without the welfare concerns and the increased time and cost associated with live animal research. Recent advances with in vitro cell culture systems utilizing human and laboratory animal tissues have been reported, but implementation of these technologies in veterinary species has been slower. Organoids are a physiologically representative 3D cell culture system that can be maintained long-term. By combining organoid culture with cryopreservation, a long-term, experimental model can be available for year-round application, thus bypassing seasonality and reproductive tract availability restrictions. Here we report the generation and cryopreservation of feline oviductal organoids for the first time. Optimal culture medium for the generation of feline oviductal organoids was established, and organoids were successfully cryopreserved using three different freezing media with organoids from each treatment demonstrating comparable viability, growth rate, and protein expression after thawing and culture. Feline oviductal organoids may facilitate an in vivo-like environment that, in conjunction with co-culture for in vitro maturation and in vitro fertilization, may positively influence in vitro gamete and embryo development, embryo quality, and pregnancy rates after embryo transfer in domestic and nondomestic felids. Furthermore, readily available cryopreserved feline oviductal organoids will facilitate this co-culture, which is of particular importance to endangered felid breeding programs where tissue and gamete samples are often opportunistically obtained with little or no notice.