Functional and molecular characterization of equine intestinal organoids across media conditions and intestinal segments

BACKGROUND: Gastrointestinal (GI) disease is a major cause of morbidity and mortality in horses, with disruption of the intestinal epithelial barrier playing a central role in disease pathogenesis. A deeper understanding of the molecular and functional properties of the equine intestinal barrier is essential to improve diagnostics and therapeutics. While intestinal organoids have emerged as a promising tool for modeling GI physiology and disease, equine-specific data remain limited. Existing studies vary in methodology and often lack functional characterization, particularly across different intestinal regions. The objective of this study was to establish a protocol for culturing equine intestinal organoids from distinct GI segments and to evaluate their barrier-related properties in comparison to native tissue. RESULTS: Organoids were successfully generated from equine duodenum, jejunum, and right dorsal colon using commercially available organoid growth (OGM) and organoid differentiation (ODM) media. All organoids formed spherical or budding structures with a central lumen and displayed viability across passages. Organoids in both media exhibited functional barrier characteristics, including transepithelial electrical resistance (TEER) and mucus production. However, transcriptomic and proteomic analysis revealed that ODM-grown organoids more closely resembled their tissue of origin than OGM-grown counterparts. Similarity was greatest in pathways related to cell adhesion, tight junctions, and epithelial transport. Discrepancies between organoids and tissue were largely related to metabolic activity and nutrient absorptive functions. Importantly, organoids retained segment-specific expression patterns, including absorptive and secretory markers, and more so in colonic organoids compared to small intestinal organoids. CONCLUSIONS: This study provides a detailed morphologic, functional, and molecular characterization of equine intestinal organoids derived from three distinct GI segments. Our findings contribute to the body of evidence demonstrating the importance of media composition to epithelial differentiation and segment-specific physiology. It also lays the groundwork for future applications, including host–pathogen interaction studies, drug permeability assays, and investigation of mucosal repair and regeneration in a segment-specific manner.