Extracorporeal bioartificial liver devices (BAL) have been in the development to help recovery from acute liver failure and provide a bridge to liver transplantation. One challenge of BAL is to maintain primary isolated hepatocytes healthy and fully functional. One of the most successful methods in retaining hepatocyte function has been their coculture with other cell types, since coculture reconstructs the cell-to-cell contacts found in vivo that are critical for intercellular signaling. In this study, we have investigated primary hepatocyte coculture with rat microvascular endothelial cells (RMEC) and primary stellate cells using a peptide based synthetic self-assembling nanostructure hydrogel. It has an average pore size of 50~200nm and promotes cell attachment. Our experimental results show that hepatocytes attached to 3 dimensional nanofibers after seeding and migrated to form stable hepatocyte spheroids within 3 to 4 days, and preseeded endothelial cells formed vascular structure to prevent nutrition delivery complexity for hepatocytes inside spheroids. In addition, extracellular matrix proteins synthesized by hepatic stellate cells (HSC) stabilize the hepatocyte spheroid structures thus maintaining the hepatocyte function for long term in vitro cultures. We observed better albumin and urea functions in the early hepatocyte spheroids culture and an improved cytochrome P450 function in this hydrogel as compared to collagen sandwich configuration. The liver tissue-like structure and maintenance of efficient liver functions demonstrated in this system suggests that the spheroids of hepatocytes cocultured with rat RMECs and HSCs are valuable in long-term toxicological studies and have great applications in bioartificial liver devices. Furthermore, because of the bio-compatibility of this synthetic hydrogel, the peptide based scaffold culture method may be of immense use in the area of hepatocyte transplantation.