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

源于组织工程概念的应用激发了人们对再生医学领域的浓厚兴趣，再生医学作为新的下一代疗法。在这样的情况下，一个小但有功能的肝组织系统可以被设计成提供与正常、功能良好的肝脏的几个百分点成比例的同等生物功能，这将有可能纠正由于各种形式的遗传性代谢缺陷造成的许多疾病表型。然而，要获得足够的肝细胞植入和持久的组织功能一直是困难的。这些功能不佳的主要原因之一是移植的肝细胞之间的细胞接触不足。最近，我们的团队创造了细胞片工程技术，使用移植了温度响应性聚合物的培养表面，允许将细胞收获为二维组织片。在本研究中，我们建立了…程序更多的e来创建统一的肝细胞片层。利用肝细胞片，我们建立了一种新的肝组织工程方法，在皮下空间建立了二维和三维的肝组织系统。方法：我们研制了碱性成纤维细胞生长因子释放装置(Am J TRANSPL 6：50,2006)，以建立带血管的皮下间隙。该装置被插入皮下间隙。几天后，当设备周围形成活跃的血运间隔时，我们移除了设备以获得血运平台。为了制作肝细胞片，将分离的肝细胞接种在培养皿中，共价嫁接有温度响应性聚合物聚(N-异丙基丙烯酰胺)(PIPAAm)。待培养的肝细胞达到融合后，将培养温度降至20℃，自发脱离PIPAAm培养皿，获得均匀连接的肝细胞薄片。将收获的组织片移植到带血管蒂皮下腔内。结果：组织学和超微结构分析表明，收获的肝细胞片含有细胞间微结构，包括胆小管、桥粒和缝隙连接，这些是显示分化的肝功能的基本特征。当我们将单个肝细胞片移植到带血管的皮下室时，工程化组织存活了235天。组织学检查证实有2-D肝组织形成。随后，我们移植了2张或2张以上的肝组织片，以确定肝组织是否会在3D中形成结构。组织学检查显示，层状组织片形成三维肝组织。与2-D肝组织相比，工程化3-D肝组织的功能更强。我们还证实了这些皮下工程肝系统在肝脏再生增殖活性、糖原合成和药物摄入以及随后的代谢方面具有肝脏特异性功能。结论：本研究描述了一种在体外建立均匀连续的肝组织片的新方法，该片可以移植到更具空间性的2-D或3-D微型肝系统。较少

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