Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

Thumbnail Image
Gabriel, Vojtech
Zdyrski, Christopher
Sahoo, Dipak
Dao, Kimberly
Bourgois-Mochel, Agnes
Atherly, Todd
Martinez, Marilyn N.
Volpe, Donna A.
Kopper, Jamie
Allenspach, Karin
Major Professor
Committee Member
Journal Title
Journal ISSN
Volume Title
MyJove Corporation
Research Projects
Organizational Units
Journal Issue
Is Version Of
Biomedical SciencesVeterinary Clinical SciencesVeterinary Diagnostic and Production Animal Medicine
The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability. Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells. The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol describes the tasks directly preceding an experiment, including in vitro validation of experimental results. Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.
This article is published as Gabriel, Vojtech, Christopher Zdyrski, Dipak K. Sahoo, Kimberly Dao, Agnes Bourgois-Mochel, Todd Atherly, Marilyn N. Martinez et al. "Canine Intestinal Organoids in a Dual-Chamber Permeable Support System." Journal of Visualized Experiments: Jove 181 (2022). DOI: 10.3791/63612. Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.