Lallemand Animal Nutrition 1 article published in JoVE Biology Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers Eloïse Mussard1,2, Corinne Lencina1, Gaëlle Boudry3, Caroline S. Achard2, Christian Klotz4, Sylvie Combes1, Martin Beaumont1 1GenPhySE, Université de Toulouse, INRAE, ENVT, 2Lallemand Animal Nutrition, 3Institut NuMeCan, INRAE, INSERM, Université de Rennes, 4FG 16: Mycotic and Parasitic Agents and Mycobacteria, Robert Koch-Institute Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.