To evaluate the survival and function of hepatocytes (HCs) on a novel three-dimensional (3D) synthetic biodegradable polymer scaffold with an intrinsic network of interconnected channels under continuous flow conditions. The authors' laboratory has investigated HC transplantation using 3D biodegradable polymers as scaffolding as an alternative approach to treatment of end-stage liver disease. Previous studies have demonstrated survival of HCs transplanted on polymer discs in peripheral tissue sites and partial correction of single enzyme liver defects. One of the major limitations has been the insufficient survival of an adequate mass of transplanted cells; this is thought to be caused by inadequate oxygen diffusion. HCs and nonparenchymal liver cells from Lewis rats were seeded onto 3D biodegradable polymer scaffolds. Microporous 3D polymers were created using 3D printing on copolymers of polylactide-coglycolide. The cell/polymer constructs were placed in static culture or continuous flow conditions. The devices were retrieved after 2 days and examined by scanning electron microscopy and histology. Culture medium was analyzed for albumin by enzyme-linked immunosorbent assay (ELISA). Differences in culture parameters including pH, PCO2, PO2, glucose, lactate, and HCO3 were examined. Scanning electron microscopy revealed successful attachment of HCs on the 3D polymer in both static and flow conditions. Histology demonstrated viable HCs in both conditions. ELISA demonstrated a significantly higher mean concentration of albumin in flow conditions than in static conditions. Culture parameter analysis revealed a significantly higher PO2 and glucose level, and a more physiologic pH in flow conditions than in static conditions. HCs cocultured with nonparenchymal cells can attach to and survive on the 3D polymer scaffolds in both static and flow conditions in the size and configuration used in this study. Flow conditions may provide a more conducive environment for HC metabolism and albumin synthesis than static conditions. The authors hypothesize that flow through directed channels will be necessary for the transfer of large masses of cells when implantation studies are initiated.