The fabrication process for a novel three-dimensional (3D) chilosan/gelatin scaffold with predefined multilevel internal architectures and highly porous structures is presented combining solid freeform fabrication (SFF), microreplication and lyophilization techniques. The computer model of the scaffold is designed with biological data such as branching angle in liver vascular cast incorporated. Stereolithography (SL), known as a SFF technique, is utilized to build the resin mould, based on which poly-dimethylsilicone (PDMS) mould is produced by microreplication. The chitosan/gelatin hybrid solution is then cast onto the PDMS mould for pre-freeze and the monolayer porous structures with organized internal morphology are produced upon lyophilization. The 3D scaffold can be constructed via stacking these monolayer structures. The properties of porous structure, such as porosity, pore size and micromorphology as well as wall thickness, were investigated. Scanning electron microscopy (SEM) demonstrated that the scaffold possesses multilevel organized internal morphologies including vascular systems (portal vein, artery and hepatic vein) and parenchymal component (hepatocyte chamber). These organized structures enable orderly arrangement of hepatocyte and hepatic nonparenchymal cells and co-culture in the same 3D scaffold to guide liver regeneration in a controlled manner. Cell culture experiment in vitro showed that hepatocytes perform better in the well-defined chitosan/gelatin scaffold than in porous scaffold. This approach makes it flexible to investigate the relationship between internal scaffold microstructure and hepatocyte behavior in vitro. It also provides a new way to fabricate complex 3D scaffold using various natural biomaterials for vital organ engineering. (c) 2007 Elsevier Ltd. All rights reserved.