ISOLATING AND MAINTAINING HIGHLY POLARIZED PRIMARY EPITHELIAL-CELLS FROM NORMAL HUMAN DUODENUM FOR GROWTH AS SPHEROID-LIKE VESICLES

A method is described for the three-dimensional (3-D) in vitro culture of nontransformed gastrointestinal epithelial cells from the human du odenal mucosa. Biopsies obtained from human duodenum were finely mince d. The tissue fragments were suspended in culture medium supplemented with 5% fetal calf serum and the appropriate antibiotics. The suspende d mucosal fragments generated spheroid-like multicellular vesicles con sisting of highly prismatic absorptive and goblet cells retaining most of the histological features of the tissue in vivo. We performed immu nocytochemical studies to determine the origin of the vesicles using m onoclonal antibodies against EP4. The histochemistry of the vesicles s howed alkaline phosphatase activity. Ultrastructural studies revealed that these cells exhibit characteristics of normal duodenal cell in vi vo: apical microvilli, glycocalyx, tight junctions and desmosomes, lat eral membrane interdigitations, mucous droplets, and a well-developed Golgi apparatus. An overgrowth of the vesicles by fibroblasts was not seen during cultivation. In contrast with the two-dimensional cell cul ture grown on artificial supports, the vesicle cell show organization similar to that of natural epithelia. The polarization and cytoarchite cture of normal gastrointestinal epithelial cells cultured as 3-D vesi cles are comparable to those known for the native tissue. This study w as undertaken to provide a morphological baseline for subsequent infec tion experiments.