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

开发 3 维肝脏组织工程程序以建立下一代疗法

基本信息

批准号：
18591957
负责人：
OHASHI Kazuo
金额：
$ 2.41万
依托单位：
Tokyo Women's Medical University (2007)Nara Medical University (2006)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

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

源自组织工程概念的应用激发了人们对再生医学领域作为新型下一代疗法的极大兴趣。在一个小的，但功能性的肝脏组织系统可以被工程化以提供与正常的，功能良好的肝脏的百分之几成比例的等效生物功能的情况下，将有可能纠正由于各种形式的遗传代谢缺陷而导致的许多疾病表型。然而，很难实现肝细胞的充分植入和持久的组织功能。这些不良功能的主要原因之一是移植肝细胞的细胞与细胞接触不足。最近，我们的小组已经创建了细胞片工程技术，使用接枝有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，我们建立了一个程序， ...更多信息 e以产生均匀的肝细胞片。在此基础上，我们建立了一种新的肝组织工程方法，在皮下构建二维和三维肝组织系统。方法：为了产生血管化的皮下隔室，我们制造了bFGF释放装置（Am J Transpl 6：50，2006）。将器械插入皮下空间。几天后，当在装置周围形成活跃的血管化隔室时，我们移除装置以获得血管化平台。为了产生肝细胞片，将分离的肝细胞铺在共价接枝有温度响应性聚合物聚（N-异丙基丙烯酰胺）（PIPAAm）的培养皿上。在平板接种的肝细胞达到汇合后，通过将培养温度降低至20 ℃从PIPAAm培养皿自发脱离，以均匀连接的组织片形式收获肝细胞。将收获的组织片移植到皮下血管化腔中。结果如下：组织学和超微结构分析显示，收获的肝细胞片含有细胞间微观结构，包括胆小管、桥粒和间隙连接，这些是表明肝脏功能分化的基本特征。当我们将单个肝细胞片移植到血管化的皮下隔室时，工程组织持续了235天。组织学检查证实了二维肝组织的形成。随后，我们移植2个或更多的肝组织片，以确定肝组织是否会形成三维结构。组织学检查显示，分层的组织片形成了三维的肝组织。与2-D肝组织相比，观察到工程化的3-D肝组织的更高的功能性。我们还证实了这些工程化皮下肝脏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的肝脏特异性功能。结论：本研究描述了一种新的方法，在体外建立一个均匀连续的肝组织片，它可以移植到一个更空间的2-D或3-D微型肝脏系统的发展。少