Generation of Intestinal Epithelial Monolayers From Single-Cell Dissociated Organoids

Intestinal organoids are generated from intestinal epithelial stem cells, forming 3D mini-guts that are often used as an in vitro model to evaluate and manipulate the regenerative capacities of intestinal epithelial stem cells. Plating 3D organoids on different substrates transforms organoids into 2D monolayers, which self-organize to form crypt-like regions (which contain stem cells and transit amplifying cells) and villus-like regions (which contain differentiated cells). This "open lumen" organization facilitates multiple biochemical and biomechanical studies that are otherwise complex in 3D organoids, such as drug applications to the cell's apical side or precise control over substrate protein composition or substrate stiffness. Here, we describe a protocol to generate homogenous intestinal monolayers from single-cell intestinal organoid suspension, resulting in de novo crypt formation. Our protocol results in higher viability of intestinal cells, allowing successful monolayer formation. Key features • This protocol requires preexisting experience in culturing mouse intestinal organoids. • This protocol requires preexisting experience in generating polyacrylamide (PAA) gels for culturing 2D monolayers. • This protocol generates intestinal monolayers that can be subjected to additional analysis, e.g., drug treatment, immunofluorescent staining, single-molecule fluorescent in-situ hybridization (smFISH), or live imaging.