Establishment of Gastrointestinal Epithelial Organoids

The intestinal epithelium constitutes a system of constant and rapid renewal triggered by proliferation of intestinal stem cells (ISCs), and is an ideal system for studying cell proliferation, migration, and differentiation. Primary cell cultures have proven to be promising for unraveling the mechanisms involved in epithelium homeostasis. In 2009, Sato et al. established a long-term primary culture to generate epithelial organoids (enteroids) with crypt- and villus-like epithelial domains representing the complete census of progenitors and differentiated cells. Similarly, isolated ISCs expressing Lgr5 (leucine-rich repeat-containing G protein-coupled receptor) can generate enteroids. Here, we describe methods to establish gastric, small intestinal, and colonic epithelial organoids and generate Lgr5(+ve) single cell-derived epithelial organoids. We also describe the imaging techniques used to characterize those organoids. This in vitro model constitutes a powerful tool for studying stem cell biology and intestinal epithelial cell physiology throughout the digestive tract. Curr. Protoc. Mouse Biol. 3:217-240 © 2013 by John Wiley & Sons, Inc.