Isolation and Co-culture of Paneth Cells and Intestinal Stem Cells

Crypts at the base of intestinal villi contain intestinal stem cells (ISCs) and Paneth cells, the latter of which work as niche cells for ISCs. When isolated and cultured in the presence of specific growth factors, crypts give rise to self-renewing 3D structures called organoids that are highly similar to the crypt-villus structure of the small intestine. However, the organoid culture from whole crypts does not allow investigators to determine the contribution of their individual components, namely ISCs and Paneth cells, to organoid formation efficiency. Here, we describe the method to isolate Paneth cells and ISCs by flow cytometry and co-culture them to form organoids. This approach allows the determination of the contribution of Paneth cells or ISCs to organoid formation and provides a novel tool to analyze the function of Paneth cells, the main component of the intestinal stem cell niche. Key features • This protocol introduces the method for isolating Paneth cells and <i>Lgr5+</i> ISCs by flow cytometry and co-culturing them. • This protocol allows analyzing the effect of genetic or biochemical modifications of Paneth or <i>Lgr5⁺</i> ISCs on organoid formation. • This protocol provides a new platform to analyze Paneth cell function.