A Preclinical Model to Assess Intestinal Barrier Integrity Using Canine Enteroids and Colonoids

Abstract

While two-dimensional (2D) cell cultures, such as Caco-2 and Madin–Darby canine kidney (MDCK) cells are widely used in a variety of biological models, these two-dimensional in vitro systems present inherent limitations in replicating the complexities of in vivo biology. Recent progress in three-dimensional organoid technology has the potential to address these limitations. In this study, the characteristics of conventional 2D cell culture systems were compared to those of canine intestinal organoids (enteroids, ENT, and colonoids, COL). Light microscopy and transmission electron microscopy were employed to evaluate the microanatomy of ENT, COL, Caco-2, and MDCK cell monolayers, while transepithelial electrical resistance (TEER) values were measured to assess monolayer integrity. The TEER values of canine ENT monolayers more closely approximated reported TEER values for human small intestines compared to Caco-2 and MDCK monolayers. Additionally, canine ENT demonstrated greater monolayer stability than Caco-2 and MDCK cells. Notably, while all systems displayed desmosomes, canine ENT and COL exclusively produced mucus. These findings highlight the potential of the canine organoid system as a more biologically relevant model for in vitro studies, addressing the limitations of conventional 2D cell culture systems.Simple Summary: Immortalized cell lines are often used to model biological systems, such as the intestinal epithelium. Compared to immortalized cell lines, which are composed of identical cell clones, organoids derived from adult stem cells may represent a more accurate biological model, since they can differentiate into specialized intestinal epithelial cell types. In this study, we isolated adult stem cells from dog intestinal samples, which can be obtained with minimally invasive methods. These adult intestinal stem cells were grown into three-dimensional organoids, which recapitulate the superficial layers (epithelium) of the original tissue. The intestinal organoids were examined under a microscope to assess their similarity to the original tissue, then cultured and compared to two immortalized cell lines. The organoids had features similar to intestinal tissue, such as mucus production. In the 2D cell culture system, the organoids formed a more consistent layer than conventional cell lines, demonstrating similar integrity to that of the human intestine. These findings suggest that organoid cultures derived from dog intestinal adult stem cells can effectively be utilized in traditional cell culture systems.