105 Generation of bovine oviductal organoids with apical-out polarity

The oviduct serves as the site of fertilization and early preimplantation embryo development in female reproduction. Oviductal epithelium includes ciliated cells to aid in gamete/embryo transport and secretory cells that contribute to the production of oviductal fluid. To better mimic the in vivo oviduct environment, development of in vitro models that maintain cellular morphology and physiological function over the long term are required. Organoids are three-dimensional culture systems that recapitulate the in vivo cell composition and can maintain long-term functionality. Organoids are generated by embedding cells into an extracellular matrix (Ultimatrix, Cultrex), with the resulting organoids forming the apical surface of the epithelial membrane facing inwards, thus making access to the apical surface and the intraluminal fluid a challenge to study. The aim of this study was to generate and culture bovine oviductal organoids with an apical-out orientation. Oviductal epithelial cells were isolated from the reproductive tracts of female beef cattle, aged 2–5 years (n = 15). These tracts were identified to be in diestrus by the presence of a corpus luteum. Isolated oviductal cells were cultured in Ultimatrix, which was then overlaid with organoid culture medium (OCM) as described previously (Menjivar et al. 2023 BMC Genom. 24, 646) and cultured for 14 days until organoids developed. After 14 days of culture, organoids (n = 50) were removed intact from the Ultimatrix and washed by centrifugation at 100g for 1 min. Unlike standard organoid culture protocols, the pelleted organoids were suspended in OCM and plated without Ultimatrix onto nontreated culture plates to reduce risk of cell adherence. To avoid organoid clumping, the suspension was agitated twice daily using a 1-mL pipette. The organoids were cultured for a total of 7–10 days with the culture medium replaced every 2 days by transferring the suspension to an Eppendorf tube and centrifugating at 100g for 3 min followed by aspiration of half the total supernatant and replaced with an equivalent amount of new OCM. The organoids were resuspended and seeded into a nontreated plate. At the end of the culture period, subsets of polarity reversed organoids were removed for the following: (1) fixed with 4% paraformaldehyde and immunofluorescently stained for Pax8 to localize the secretory cells, Acytub to identify cilia, and DAPI to identify nuclei of viable cells; and (2) fixed for transmission electron microscopy (TEM) to identify anatomical features and morphology. Immunofluorescent staining confirmed both ciliated and secretory cells were present in all the organoids sampled, verifying that reversed polarity organoids recapitulate in vivo oviductal cellular diversity and localization. TEM images confirmed that apical-out polarity of the organoids was achieved, with microvilli visible on the external surface and the basement membrane facing the organoid lumen. Multivesicular structures suggestive of extracellular vesicles were also identified on TEM. This study will provide data for reversed polarity of bovine oviductal organoids, which enables studies for maternal-embryo communication under in vitro or co-culture conditions.