Development of 3-dimensional liver tissue engineering procedure toward establishment of next generation therapies

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The application derived from the concept of tissue engineering have spurred significant interest into the filed of regenerative medicine as novel, next generation therapies. Under circumstances in which a small, but functional liver tissue system could be engineered to provide the equivalent biological function proportional to a few percent of a normal, well-functioning liver, it would be possible to correct many disease phenotypes as a result of various forms of inherited metabolic deficiencies. However, it has been difficult to achieve sufficient engraftment of hepatocytes and persistent tissue functions. One of the major reasons for these poor functionalities has been insufficient cell-to-cell contact of the engrafted hepatocytes. Recently, our group has created cell sheet engineering technologies using culture surfaces grafted with the temperature-responsive polymer that allows for harvesting the cells as a 2-dimensional tissue sheet. In the present study, we established a procedur … More e to create uniform hepatocyte sheet. Using the hepatocyte sheet, we then established a novel liver tissue engineering approach to create 2-dimsneional and 3-dimensional liver tissue system in the subcutaneous space. Methods: In order to create vascularized subcutaneous compartments, we created bFGF-releasing device (Am J Transpl 6: 50, 2006) . The device was inserted into the subcutaneous space. The days later, at a time when active vascularized compartments were formed around the device, we removed the device to obtain vascularized platform. To create hepatocyte sheets, isolated hepaotcytes were plated on culture dishes covalently grafted with the temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) . After the plated hepatocytes reached confluency, hepatocytes were harvested as a uniformly connected tissue-sheet by spontaneous detachment from the PIPAAm dishes by lowering the culture temperature to 20C. The harvested tissue sheets were transplanted into the subcutaneous vascularized cavity. Results: Histological and ultrastructural analyses revealed that the harvested hepatocyte sheets contained intercellular microstructures including bile canaliculi, desmosomes, and gap junctions, which are essential traits that demonstrate differentiated hepatic functions. When we transplant single hepatocyte sheets into the vascularized subcutaneous compartment, the engineered tissues persisted for 235 days. Histological examination confirmed the formation of 2-D hepatic tissues. Subsequently, we transplanted 2 or more hepatic tissue sheets to determine whether the liver tissues would form structure in 3-D. Histological examination revealed that the layered tissues sheets formed 3-D hepatic tissues. Higher functionality of the engineered 3-D hepatic tissues were observed compared to 2-D hepatic tissues. We have also confirmed the liver-specific functions of these engineered subcutaneous liver system in terms of liver regenerative proliferation activity, glycogen synthesis, and drug intake followed by their metabolism. Conclusions: The present study describes a novel approach to create a uniformly continuous sheet of hepatic tissue in vitro, which can be transplanted to develop into a more spatial 2-D or 3-D miniature liver system. Less