Liver tissue constructs with excretory function are crucial to developing realistic hepatocyte models for engineering effective bioartificial liver-assisted devices and for modeling the in vivo tissue. Current hepatocyte in vitro models suffer from limited or inefficient hepatocyte repolarization, which results in poor removal of xenobiotics and other waste products from the cells. We hypothesized that the temporal and spatial presentation of the cell matrix and cell-cell contacts as polarity cues would be important to define the axis of polarization to improve the excretory function of hepatocytes. The spatial presentation of polarity cues can be best achieved with sandwich configuration. We improve the temporal presentation of polarity cues by introducing the collagen overlay immediately in synchrony with cell-cell contacts instead of after 24 h in conventional sandwich culture. We demonstrate that the immediate presentation of the collagen matrix overlay enhances the formation of apicobasolateral domains, tight junctions, and the recovery of the functional activity of 2 canalicular transporters, the multidrug resistance-associated protein (Mrp2) and P-glycoprotein (P-gp) at 48 h of culture, and enhances the albumin secretion, urea production, and 7-ethoxyresorufin-O-deethylation cytochrome P450 activities of hepatocytes over 14 days of culture as compared to the 24-h overlay controls. The improvement in the excretory function of hepatocytes for the removal of waste products deleterious to cells may improve the functional maintenance and the in vivo fidelity of tissue-engineered liver constructs.