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.

Center for Experimental Medicine Institute for Clinical and Experimental Medicine 14021 Prague Czech Republic

D Health Solutions Inc Athens GA 30602 USA

Department of Animal Medicine and Surgery College of Veterinary Medicine Complutense University of Madrid 28040 Madrid Spain

Department of Biomedical Sciences Iowa State University Ames IA 50011 USA

Department of Pathology Carver College of Medicine University of Iowa Iowa City IA 52242 USA

Department of Pathology College of Veterinary Medicine University of Georgia Athens GA 30602 USA

Department of Pathology Precision One Health Initiative College of Veterinary Medicine University of Georgia Athens GA 30602 USA

Department of Veterinary Clinical Sciences Iowa State University Ames IA 50011 USA

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