Conventional in-vitro models of intestinal epithelium such as human intestinal/colonic cancer cell lines lack essential features of the canine intestine, limiting their use to study canine intestinal barrier function, cellular metabolism, or host-microbe interactions. Intestinal organoids, also known as enteroids, represent a recent primary cell culture technology that has revolutionized the field of intestinal disease and physiology modeling. Enteroids are 3D structures that are composed of a single layer epithelium arranged in crypt- and villi-like domains with its apical and basal membranes facing the luminal and external sides of the enteroid, respectively. The conversion of the 3D organization of enteroids into a 2D monolayer enables accessibility to intestinal cells' apical membrane for experimental infections or drug treatments. This study aimed to develop a canine enteroid-derived monolayer model to study gut barrier structure and function. Jejunal segments were obtained from three donor dogs during a resection and anastomosis surgery and used to isolate the intestinal crypts by EDTA-based tissue dissociation. Then, the intestinal crypts were resuspended in growth factor reduced Matrigel and supplemented with the commercial IntestiCultâ„¢ Human Organoid Growth Medium to generate 3D enteroids. Canine enteroids were mechanically and enzymatically disrupted into single cells and cultured on Matrigel-coated porous membranes of transwell culture systems, reaching 100% confluency around day four. We performed immunofluorescence, histochemistry, and transmission electron microscopy (TEM) to study intestinal epithelium morphology and structure. Intestinal epithelium barrier function was analyzed by Fluorescein Isothiocyanate-Dextran 4kD (FITC-D4) intestinal permeability Assay and Trans-Epithelial Electrical Resistance (TEER) measurement. Canine enteroids and 2D monolayers showed positive staining for ZO-1, Ki67, and Periodic Acid of Schiff (PAS), indicating epithelial cell heterogeneity and polarization. A functional intestinal barrier was confirmed by FITC-D4 assay and TEER measurement. This protocol provides a canine-specific, complex, and long-term ex-vivo model to study intestinal epithelial function.
