BASEMENT-MEMBRANE FORMATION OF FETAL MOUSE INTESTINAL EPITHELIAL-CELLS IN ORGANOID CULTURES

Basement membrane formation of fetal mouse intestinal epithelial cells was investigated in organoid cultures. Intestinal cells were dissocia ted with a commercial collagenase/dispase preparation, and the cells w ere grown at high density on a membrane filter at the interface betwee n the medium and air. This type of culture allows the histotypical reo rganization of cells. After 2 days in culture, epithelial cells began to accumulate on the surface, in particular at the periphery of the cu lture. These cells were usually cuboid, and small vesicles were formed in the center of the culture. Laminin-positive material was observed at peripheral sites. However, no basement membrane could be identified beneath the epithelial cells at the electron-microscopic level. After 3 days, epithelial cells that had gathered at the periphery became co lumnar in shape. Laminin-positive material extended across the surface of the culture. However, the vesicles formed in the center of the cul ture were not associated with laminin-positive material. Basement memb rane was observed by electron microscopy at some sites beneath groups of epithelial cells, but did not extend continuously beneath these cel ls. Some epithelial cells made contact with the underlying mesenchymal cells through the discontinuous basement membrane via intercellular c ontacts. After 5-6 days, the surface of tile culture was almost comple tely covered with epithelial cells and, at some sites, villus-like str uctures were visible. Laminin-positive material was clearly detectable under epithelial cells, as well as around epithelial vesicles located in the center of the culture, By electron microscopy, basement membra ne was clearly visible between the epithelial and mesenchymal cells. A fter 9 days, villus-like structures were rarely observed. After 3 week s, the cell mass had become smaller and villi had disappeared. Basemen t membrane was extensively folded and no basement membrane was visible at some sites. Formation of basement membrane by epithelial cells in monolayer culture occurs in an incomplete and irregular manner. It occ urs rapidly in organoid cultures that include mesenchyme and epitheliu m. The organoid culture used here should be a useful tool for studies of the formation and degeneration of the basement membrane as well as interactions between the epithelium and mesenchyme.