Center for Veterinary Medicine, Food and Drug Administration 1 article published in JoVE Immunology and Infection Canine Intestinal Organoids in a Dual-Chamber Permeable Support System Vojtech Gabriel1, Christopher Zdyrski1, Dipak K. Sahoo2, Kimberly Dao3, Agnes Bourgois-Mochel2, Todd Atherly3, Marilyn N. Martinez4, Donna A. Volpe5, Jamie Kopper2, Karin Allenspach2,3, Jonathan P. Mochel1,3 1Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, 33D Health Solutions Inc., 4Office of New Animal Drug Evaluation, Center for Veterinary Medicine, Food and Drug Administration, 5Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.