Primary culture and transfection of epithelial cells of human small intestine

Background: So far, no techniques are available for primary culture and efficient transfection of human small-intestinal enterocytes, which would provide a valuable tool to investigate intestinal function. Methods: Human small-intestinal biopsy specimens were treated with collagenase and dispase. Resulting crypt units were cultured for several days. Using the intestinal epithelial cell lines Caco-2 and HT-29, we established optimal conditions for transfection of a control plasmid, which were then applied to primary cultured cells. Results: Cells growing out of crypt units formed monolayer-like sheets and proliferated for several days. Most of the cells could be stained with antibodies against epithelial markers. Among seven different transfection reagents tested, Lipofectamine was the most potent, with transfection efficiencies up to 25% for primary enterocytes. Conclusions: An easy technique was developed providing viable small-intestinal enterocytes that can be efficiently transfected.