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

Abstract

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.