Hepatocyte transplantation is being explored as a treatment strategy for end-stage liver disease; however, the main limitation is the insufficient vascularization of transplanted hepatocytes. To overcome this problem, a suitable 3D microenvironment and the types of transplanted cells must be considered for hepatocyte transplantation. In this study, a growth factor (GF)/heparin-immobilized collagen gel-filled polyurethane foam (PUF) scaffold was developed for angiogenesis induction and hepatocyte transplantation. First, a vascular endothelial growth factor (VEGF)/heparin-immobilized, collagen-gel-filled PUF scaffold was developed to establish a prevascularized cavity in the subcutaneous space in rats. Second, accompanied by 70% partial hepatectomy (PH), hepatocytes were embedded inside heparin-immobilized, collagen-gel-filled PUF scaffolds, and were transplanted into the VEGF-induced prevascularized cavity. The benefits of using this system were confirmed by using three types of hepatocytes, namely single hepatocyte, hepatocyte spheroids, and fetal hepatocytes. The normalized hemoglobin content and live nucleus numbers were determined separately to evaluate the angiogenesis and viability of transplanted hepatocytes. In summary, after PH pretreatment, transplantation of fetal hepatocyte-embedded, heparin-immobilized, collagen-gel-filled PUF scaffold into a VEGF-induced prevascularized cavity appears to be a promising strategy for future liver tissue engineering. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.