Basement Membrane Formation of Fetal Mouse Intestinal Epithelial Cells in Organoid Cultures

Abstract

Basement membrane formation of fetal mouse intestinal epithelial cells was investigated in organoid cultures. Intestinal cells were dissociated with a commercial collagenase/dispase preparation, and the cells were grown at high density on a membrane filter at the interface between the medium and air. This type of culture allows the histotypical reorganization of cells. After 2 days in culture, epithelial cells began to accumulate on the surface, in particular at the periphery of the culture. 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 columnar in shape. Laminin-positive material extended across the surface of the culture. However, the vesicles formed in the center of the culture were not associated with laminin-positive material. Basement membrane was observed by electron microscopy at some sites beneath groups of epithelial cells, but did not extend continuously beneath these cells. Some epithelial cells made contact with the underlying mesenchymal cells through the discontinuous basement membrane via intercellular contacts. After 5-6 days, the surface of the culture was almost completely covered with epithelial cells and, at some sites, villus-like structures 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 membrane was clearly visible between the epithelial and mesenchymal cells. After 9 days, villus-like structures were rarely observed. After 3 weeks, the cell mass had become smaller and villi had disappeared. Basement 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 occurs rapidly in organoid cultures that include mesenchyme and epithelium. 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.

Reorganized small intestine from fetal mouse as an in vitro wound healing model

“…Day 0 of gestation was defined as the day of vaginal plug appearance. The cell isolation and culture methods used were as described by Murata et al 12 Briefly, 30mm of small intestine, from the duodeBackground. We developed a method for reorganizing the mouse small intestine.…”

“…In the monolayer culture, epithelium formation was not observed, and only fibroblasts were stratified on the bottom of the culture dishes. However, epithelium formation was observed in the organoid culture, as described by Murata et al 12 Therefore, in the present study, we used this culture method in an attempt to elucidate the mechanism of epithelial development and repair.…”

“…10 However, with the introduction of collagen-coated plates, some progress was made in this field. 11 Murata et al 12 successfully formed a reorganized small intestine using a highdensity cell culture (organoid culture) in which the small intestine of fetal mouse was dispersed into single cells. Fujimoto 13 compared the reorganization of epithelium in monolayer and organoid cultures.…”