Conventional two-dimensional cultures in monolayer and sandwich configuration have been used as a model for in vitro drug testing. However, these culture configurations do not present the actual in vivo liver cytoarchitecture for the hepatocytes cultures and thus they may compromise the cells liver-specific functions and their cuboidal morphology over longer term culture. In this study, we present a three-dimensional polydimethylsiloxane (PDMS) scaffold with interconnected spherical macropores for the culturing of rat liver cells (hepatocytes). The scaffolds were integrated into our perfusion enhanced bioreactor to improve the nutrients and gas supply for cell cultures. The liver-specific functions of the cell culture were assessed by their albumin and urea production, and the changes in the cell morphology were tracked by immunofluorescence staining over 9 days of culture period. N-Acetyl-Para-Amino-Phenol (acetaminophen) was used as drug model to investigate the response of cells to drug in our scaffold-bioreactor system. Our experimental results revealed that the perfusion enhanced PDMS-based scaffold system provides a more conducive microenvironment with better cell-to-cell contacts among the hepatocytes that maintains the culture specific enzymatic functions and their cuboidal morphology during the culturing period. The numerical simulation results further showed improved oxygen distribution within the culturing chamber with the scaffold providing an additional function of shielding the cell cultures from the potentially detrimental fluid induced shear stresses. In conclusion, this study could serve a crucial role as a platform for future preclinical hepatotoxicity testing. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:418-428, 2014