Protocol for a primary human 2D intestinal epithelium-dendritic cell co-culture organoid model to study mucosal immunity and viral infections.

The intestinal mucosa is a major HIV-1 infection site, making immune-augmented intestinal models essential for mechanistic and therapeutic studies. We present a protocol that converts human intestinal organoids into polarized 2D epithelial monolayers and incorporates basolateral dendritic cells to create a biomimetic intestinal mucosal model. We describe steps for monolayer formation, integrity assessment, immune cell co-culture, virus inoculation, and analyses by flow cytometry and confocal microscopy. This immuno-organoid platform enables controlled HIV-1 exposure with immunological and virological readouts. For complete details on the use and execution of this protocol, please refer to Rader et al.1.