Xenotransplantation of fetal porcine hepatocytes in rats using a tissue engineering approach

Hepatocytes can be successfully transplanted into highly vascular sites such as the spleen, liver, and lungs. Subcutaneous sites lack adequate vascularization to nutritionally support transplanted hepatocytes. We recently reported that matrix-immobilized angiogenic growth factors, e.g., endothelial cell growth factor (ECGF), can induce a high degree of neovascularization. Using this technique, we explored the possibility of transplanting isolated fetal porcine hepatocytes to create liver tissue organoids at a specific subcutaneous site. We evaluated chitosan as a scaffold biomaterial because of its structural similarity to glycosaminoglycans; glycosaminoglycans play a critical role in cell attachment, differentiation, and morphogenesis. Freshly isolated fetal porcine hepatocytes (FPH) (viability greater than 97%) were cultured on modified chitosan scaffolds and transplanted into rat groin fat pads with or without ECGF-induced neovascularization. Cell density and attachment kinetics on chitosan were examined by scanning electron microscopy (SEM) and quantified using a flavianic acid binding assay. Hepatocyte viability and liver organoid formation were examined immunohistochemically. FPH transplanted without prior neovascularization died within 1 day posttransplantation. When transplanted after ECGF-induced neovascularization, FPH thrived for at least 2 weeks and formed liver tissue like structures. Immunohistochemical analysis revealed the presence of hepatocyte-specific cytokeratin staining as well as the presence of alpha-fetoprotein. Light microscopy and SEM revealed that FPH did not change their morphology after attachment to the chitosan surfaces. Thus, chitosan-based biomaterial surfaces have good hepatocyte attachment properties, However, extensive neovascularization is essential for hepatocyte survival and organoid formation. In the future, chitosan-based biomaterials may be useful as scaffolds for creating liver tissue organoids.