The in vitro long-term expansion of primary intestinal epithelial cells has been hampered by the inability to maintain an immature stem cell population. Recent technical advances have led to the development of a novel in vitro culture system that can sustain intestinal stem cells (ISCs) using growth factors that mimic the intestinal microenvironment in combination with a three-dimensional (3D) culture. The resulting intestinal organoids display a crypt-villus architecture that recapitulates the native intestinal epithelium. Here, we describe our method for the long-term culture of intestinal epithelial organoids via consistent passaging using a gentle cell dissociation reagent to easily break the organoid into smaller pieces. The long-term cryopreservation and defining characteristics of these intestinal organoids also make this work relevant for the advancement of epithelial organoid-based therapeutic technologies by allowing the production of large numbers of cells for use in clinical applications.
Keywords: Crypt isolation; Intestinal epithelial cell; Intestine tissue; Lgr5; Long-term culture; Organoid.