Updated Protocols for 3D and 2D Culture and Histological Analysis of Intestinal Organoids

The intestinal epithelium maintains continuous self-renewal and multi-differentiation capacities through the integrated regulation of multiple signaling pathways, including Wnt, bone morphogenetic protein, epidermal growth factor (EGF), and Notch signaling pathways. Based on this principle, a defined combination of EGF, Noggin, and the Wnt agonist R-spondin has been shown to support long-term expansion of mouse intestinal stem cells and the formation of organoids with indefinite self-renewal and full differentiation capacity. In human intestinal organoid cultures, the addition of small molecule inhibitors targeting p38 and TGF-β pathways facilitated epithelial propagation, while replacement of EGF and a p38 inhibitor with insulin-like growth factor-1 and fibroblast growth factor-2 enabled efficient multilineage differentiation. Recently, the monolayer culture of intestinal organoid has been increasingly used due to its advantages of direct access to the apical membrane and its ability to co-culture with gut microbiota. Here, we summarize our recent technological advances in human intestinal organoid culture and histological analysis, providing updated protocols that will deepen the understanding of intestinal homeostasis and the pathogenesis of intestinal diseases.