Protocol for generating iPSC-derived intestinal organoids through dissection to model cystic fibrosis therapies

Intestinal organoids (IOs) are versatile, physiologically relevant models of the human gut. In cystic fibrosis research, IOs help evaluate CFTR correctors, potentiators, and translational readthrough compounds. Here, we present a protocol for generating induced pluripotent stem cell (iPSC)-derived IOs with high phenotypic similarity to primary material. We describe steps for establishing iPSC culture, generating intestinal progenitors via directed differentiation, and maturing these intestinal progenitors using three-dimensional culture. We then detail procedures for establishing intestinal organoid cultures from these progenitors using dissection-based techniques.