Hepatocytes within current bioartificial liver designs are unable to maintain the wide range of differentiated functions observed for the liver in vivo. As recent studies suggest, the absence of controlled interactions between hepatocytes and non-parenchymal cell populations of the liver may contribute to this hepatocyte dedifferentiation. The current study investigates the effect of Kupffer cells, fibroblasts, and human umbilical vein endothelial cells on hepatocyte function. To effectively study the effect of these heterotypic cell-to-cell interactions, it is necessary to establish a culture environment that controls the interactions between multiple cell types. Micropatterning is such a technique and was used in the current study. In addition, to elucidate the influence of soluble factors on hepatocyte function, the micropatterned results were compared with those of the trans-well culture system. Specifically, we compared the morphological and functional changes of hepatocytes cultured with these cells at varying ratios. Our results suggest that direct heterotypic cell-to-cell contact between hepatocytes and fibroblasts, and soluble factor exchanges between hepatocytes and human umbilical vein endothelial cells, significantly enhanced hepatocyte performance.