Abstract
Intestinal organoid culture is a powerful tool to model stem and epithelial cell biology. Here we present a protocol to generate long-lived two-dimensional monolayers of all major intestinal epithelial cell types using primary mouse colon stem cells grown under air-liquid interface. An advantage of this protocol over conventional 3D organoid culture is that the monolayer is self-renewing for at least four weeks without passaging, allowing long-term studies of intestinal development and response to injury or challenge. Mouse colonic stem cells are first expanded in a conditioned medium containing Wnt, R-spondin, and Noggin. The stem cells are then seeded on a semi-permeable membrane to form a continuous monolayer. After seven days of submerged cell growth, the monolayer is exposed to an Air-liquid interface (ALI) by removing conditioned media from the apical compartment. This results in epithelial differentiation and formation of numerous self-organizing proliferative foci that resemble “flattened” colonic crypts. Stem cells and differentiated lineages co-exist in this monolayer for at least four weeks. We further demonstrate the ability to model injury-repair cycles by re-submerging the cells under conditioned media, which leads to a loss of differentiated cells while sustaining the regenerative stem cells. The differentiating monolayer can then be re-established by resuming the Air-liquid interface. In this protocol, we additionally present methods for histological analysis including paraffin embedding and whole mount imaging. This monolayer system can be adapted to study many aspects of long-term intestinal development, including stem cell dynamics, host-pathogen interactions, and metabolism.
