Intestinal organoids (enteroids) are three-dimensional (3D) in vitro cellular structures processing all epithelial cell populations, including stem cells, enterocytes, goblet cells, enteroendocrine cells, Paneth cells, and Tuft cells. Over the last few decades, a significant amount of work has been put into developing isolation and culture conditions of enteroids from multiple species at different stages of their development. Two major types of enteroids, basolateral out and apical out, have been established to facilitate research interests with serosal and luminal manipulation, respectively. However, as an ex vivo cellular model that recapitulates the in vivo conditions of the intestine, the functional analyses of these enteroids have not been thoroughly investigated.

A variety of researchers will benefit from establishing repeatable and reliable functional evaluation protocols in order to characterize the specific cells of interest in the enteroids culture, as well as to evaluate the overall enteroids culture as a mini-gut organ. The goal of this Methods Collection is to bring together the cutting-edge protocols of functional assays of the enteroids research model, including intestinal epithelial stem cell proliferation and differentiation efficiency, intestinal barrier function, inflammatory and oxidative challenge conditions, epithelial nutrient transport, nutrient sensing, goblet cell mucin secretion, intestinal hormones secretion, as well as enteroids interaction with other biological systems, such as microbiota, immune and enteric nervous system.