Generation of small intestinal organoids for experimental intestinal physiology.

Human intestinal organoids (HIOs) derived from pluripotent stem cells were first described almost a decade ago as a method to differentiate intestinal tissue containing both epithelium and supporting mesenchymal cells. The original protocol documents a directed differentiation approach to first induce definitive endoderm from pluripotent stem cells, followed by hindgut specification, resulting in the self-organization of 3D hindgut spheroids. These hindgut spheroids are then embedded in a basement membrane extracellular matrix (ECM) such as Matrigel and mature into HIOs over about 4 weeks in culture. Since the initial HIO protocol was published, the methods to generate HIOs have been updated over time including revisions to the directed differentiation protocol and implementation of new culture methods for spheroids such as embedding in alginate or polyethylene glycol hydrogels as defined alternatives to Matrigel. Additionally, HIOs have been utilized for new applications such as co-culture with bacteria. This protocol compiles the most up to date information on HIO generation and presents alternative experimental applications.