Mouse Small Intestinal Organoid Cultures.

The intestinal epithelium is a highly dynamic and self-renewing tissue that is crucial for maintaining gut homeostasis. It can be cultured in vitro from isolated crypts to form three-dimensional (3D) intestinal organoids. These organoids have the ability to proliferate and differentiate into various epithelial cell lineages, offering a more physiologically relevant model compared to traditional two-dimensional (2D) culture systems. Mesenchymal cells, located near epithelial cells, regulate epithelial behavior through paracrine signaling and provide structural support. Building on recent advances in the biology of epithelial and mesenchymal cells, we have developed a coculture system that integrates intestinal organoids with mesenchymal cells. In this system, intestinal organoids are cultured in direct or indirect contact with mesenchymal cells, allowing for the simulation of signal exchange and interactions within the in vivo-like microenvironment. This coculture system not only preserves the 3D architecture of the organoids but also enhances their physiological relevance by introducing cellular complexity. The system is capable of long-term maintenance and is adaptable to a wide range of experimental manipulations. As such, this coculture model serves as a powerful tool for studying the interactions between the intestinal epithelium and its surrounding stroma, providing new insights into stem cell biology, tissue regeneration, and disease mechanisms. Here, we introduce the methods of mouse crypt isolation, intestinal organoid culture, and its coculture with mesenchymal cell.