Current in vitro cell culture models do not reflect human physiology, and various efforts have been made to enhance existing models. Reconstitution of three-dimensional (3D) tissue structure has been one of the strategies, since 3D tissue structure provides essential cellular environmental cues for cell functions. Previously, we developed a novel hydrogel microfabrication technique for constructing an accurate 3D replica of human intestinal villi epithelium. In this study, genetic and physiological properties of the 3D villi model were examined to gain a better insight into the barrier function of gut epithelium and its interaction with microbes. Gene expression study of Caco-2 on the 3D villi scaffold revealed that expression of MUC17, which is one of the transmembrane mucins, was highly enhanced in the 3D villi model, compared to a monolayer culture. Cells on the scaffold were almost immune to bacterial infection, while MUC17 knockdown in Caco-2 cells restored bacterial infectivity. The 3D villi model also exhibited changes in the barrier function compared to the 2D model, manifested by changes in transepithelial electrical resistance (TEER) and permeability of FITC-dextran. Knockdown of MUC17 resulted in reduction of tight junction protein expression and further increase in permeability, suggesting an important role of MUC17 in the barrier function against pathogens and xenobiotics. Our study suggests that mimicking the 3D tissue architecture of the small intestine induces physiological changes in human intestinal cells.

Physiological and morphological properties of the human intestine cannot be accurately mimicked in conventional culture devices such as well plates and petri dishes where intestinal epithelial cells form a monolayer with loose contacts among cells. Here, we report a novel microfluidic cell culture device (μFCCD) that can be used to culture cells as a human intestinal model. This device enables intestinal epithelial cells (Caco-2) to grow three-dimensionally on a porous membrane coated with fibronectin between two polydimethylsiloxane (PDMS) layers. Within 3 days, Caco-2 cells cultured in the μFCCD formed villi- and crypt-like structures with small intercellular spaces, while individual cells were tightly connected to one another through the expression of the tight junction protein occludin, and were covered with a secreted mucin, MUC-2. Caco-2 cells cultured in the μFCCD for 3 days were less susceptible to bacterial attack than those cultured in transwell plates for 21 days. μFCCD-cultured Caco-2 cells also displayed physiologically relevant absorption and paracellular transport properties. These results suggest that our intestinal model more accurately mimics the morphological and physiological properties of the intestine in vivo than the conventional transwell culture model.

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