Intestinal crypt-derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus

The role of enteric neurons in driving intestinal peristalsis has been known for over a century. However, in recent decades, scientists have begun to unravel additional complex interactions between this nerve plexus and other cell populations in the intestine. Investigations into these potential interactions are complicated by a paucity of tractable models of these cellular relationships. Here, we describe a novel technique for ex vivo coculture of enteroids, so called “mini-guts,” in juxtaposition to the longitudinal muscle myenteric plexus (LMMP). Key to this system, we developed a LMMP culture media that: (i) allows the LMMP to maintain ex vivo peristalsis for 2 weeks along with proliferation of neurons, glia, smooth muscle and fibroblast cells, and (ii) supports the proliferation and differentiation of the intestinal stem cells into enteroids complete with epithelial enterocytes, Paneth cells, goblet cells, and enteroendocrine cells. Importantly, this technique identifies a culture condition that supports both the metabolic needs of intestinal epithelium as well as neuronal elements, demonstrating the feasibility of maintaining these two populations in a single culture system. This sets the stage for experiments to better define the regulatory interactions of these two important intestinal cell populations.