The generation of functionally differentiated, three-dimensional hepatic tissue from two-dimensional sheets of progenitor small hepatocytes and nonparenchymal cells

Background. The authors' laboratory has investigated tissue engineering of the liver as a novel approach for treating end-stage liver disease. Fabrication of thick, viable, three-dimensional liver tissue is limited by the lack of vascularity in the tissue-engineered constructs. To overcome this limitation, the authors fabricated three-dimensional, vascularized liver tissue in vivo from two-dimensional cell sheets created from small hepatocytes (SHC) and nonparenchymal cells (NPC) implanted into rat omentum. Methods. SHC and NPC were cultured on a silicon wafer and lifted as monolayer cell sheets. After maximal hepatotrophic stimulation was induced in the host by injecting retrorsine, creating a portacaval shunt, and performing a partial hepatectomy, these sheets were placed onto the omentum and then rolled into a three-dimensional cylinder. Results. New tissue consisting of both hepatocytes and bile duct-like structures formed by 2 weeks, and the mass of hepatocytes increased in size up to 2 months. The hepatocytes in these constructs were immunohistochemically positive for albumin and transferrin, and bile duct-like structures were positive for gamma-glutamyl transpeptidase, which suggests that they possess liver-specific function. Electron microscopy also revealed structures resembling bile canaliculi. Conclusions. Functional, morphologically complex new tissue was generated from morphologically simple monolayer cell sheets of SHC and NPC. These results represent an essential step toward the design of tissue-engineered complex vascularized thick tissue.