Behavior of fetal intestinal organ culture explanted onto a collagen substratum

Abstract

A model of organ culture of 18 day old fetal rat intestine (Quaroni, 1985) was modified and characterized in the present work with the purpose of developing an in vitro model for the study of intestinal epithelial cell behaviour. Fragments of this intestine were kept in suspension culture for 7 days and then explanted onto collagen (type I) matrix. Within a day, the fragments became anchored to the substratum and a circular monolayer grew out to about 1 cm diameter. In the fragments, an outer layer of absorptive epithelial cells came to enclose a stroma, which was polarized into a loose (mesenchymal) and a dense portion. The dense portion contained a mixture of smooth muscle cells and primitive stem-type epithelial cells ('p-cells'). After explantation, at the contact point with the matrix, the epithelium broke up and the mesenchyme grew into the matrix and anchored the fragment. The epithelial edges now became continuous with the developing monolayer. Radioautography with tritiated thymidine indicated a constant cell renewal in epithelium and monolayer apparently from foci of p-cells, a reserve population of which was seen to be sequestered among the smooth muscle cells. Activated stem cells could differentiate into three mature epithelial phenotypes, each differentiation pathway apparently being determined by the type of underlying stroma. Immunohistochemistry using gold- and fluorescein-labeled monoclonal antibodies indicated that adult differentiation-specific markers (e.g. brush border enzymes) were present in the fragment epithelium but not in the monolayer cells. On the other hand, the monolayer cells could be induced to express some of these markers by contact with mesenchymal cells or by co-culturing with fibroblastic cell lines. Matrigel substratum mixed with collagen (type I) supported the appearance in monolayer of strands positive for amino-peptidase and lactase. The model thus appears to be suitable for the in vitro study of epithelial renewal and differentiation, and it has already provided some results in this respect.