The epithelium of the small intestinal mucosa is a highly dynamic system particularly well suited for analyzing key biological phenomena such as cell differentiation and migration, cell-matrix interactions, and apoptosis. However, in vitro models of fully differentiated normal human enterocytes are still lacking. The objective of the present study was to investigate the possibility of generating such differentiated intestinal cell cultures from the fetal small intestine. For this purpose, various dissociation methods were tested in order to obtain pure, viable, and functional enterocytes. One of these methods, based on the procedure to recover epithelial cells grown on Matrigel and involving the use of Matrisperse, a nonenzymatic dissociating solution, was found to allow the isolation of the integral epithelial lining from the mesenchyme. In culture, these epithelial fractions plated on collagen I spread rapidly and reached confluence after 3-4 days. When tested after 5-7 days, these primary cultures of differentiated enterocytes (PCDE) remained well preserved. Both goblet and absorptive cells exhibit all the main characteristics of intact villus intestinal cells as assessed by electron microscopy. Indirect immunofluorescence and Western blot analyses confirmed the purity of PCDE. The functional status of these cells was demonstrated by the presence of uniformly distributed tight junction, zonula adherens, and desmosomal components at the region of cell to cell contact as well as expression of various brush border enzymes, namely sucrase-isomaltase and lactase, and goblet cell mucins. As expected, cell proliferation was found to be negligible as assessed by DNA synthesis. Taken together, these data show that primary cultures of pure and viable differentiated enterocytes can be generated from the human fetal small intestine.
Copyright 1998 Academic Press.