An Inverted Organoid-Derived Intestinal Epithelial Monolayer Model for Studying Immune Cell–Epithelial Interactions

Human intestinal organoids provide a powerful model system to study epithelial biology by preserving key characteristics of intestinal tissue organization, cellular differentiation, and function. However, their three-dimensional architecture limits direct experimental access to the apical surface, which is enclosed within the organoid lumen. This restricted accessibility complicates studies requiring controlled stimulation of epithelial compartments, barrier analysis, and investigation of interactions between epithelial and immune cells. This protocol describes the generation of an inverted organoid-derived intestinal epithelial model designed to improve accessibility to both the apical and basal compartments while maintaining essential epithelial properties. In this approach, organoid-derived single cells are seeded onto an inverted permeable support, where they establish a polarized epithelial monolayer that retains important features of intestinal differentiation and organization. The open configuration enables direct use of epithelial compartments and facilitates functional applications, including compartment-specific stimulation and immune-epithelial interaction studies. Furthermore, the model allows the integration of immune cell co-culture approaches to investigate cellular communication and migration processes. This platform provides a practical and adaptable experimental system that bridges conventional three-dimensional organoid cultures and accessible epithelial monolayer models. It offers a valuable approach for studying intestinal barrier function, inflammatory responses, immune interactions, and disease-associated mechanisms under controlled experimental conditions.