三维动态仿生微环境体外构建微肝组织的研究

Biofabrication of transplantable liver tissues/organs in vitro is one of the most promising approaches to treatment of acute liver failure and other end-stage liver diseases. In this study, we aim to fabricate transplantable liver microtissues with mature liver functions in a flow-based, three-dimensional (3D) biomimetic microenvironment, and to elucidate the underlying mechanism of biofabricating liver microtissues from induced pluripotent stem cells (iPSCs). First, human iPSCs will be induced to differentiate into hepatic cells (iPS-Heps) and supporting cells (vascular endothelial cells, iPS-Ends) using biochemical factors in vitro, respectively. Second, hexagonal cell-loaded hydrogel scaffolds (biomimetic liver lobule) will be generated using a micropatterning approach. Third, flow-based 3D microenvironment will be constructed for the co-culture of iPS-Heps and iPS-Ends with optimization on stiffness and pore structure of hydrogel scaffolds, biochemical factors and fluidic conditions in a microfluidics-based bioreactor. Lastly, liver microtissues with mature liver functions will be transplanted to a mouse model with acute liver failure. Our systematic study from microenvironment to cellular behavior, generation of liver microtissues and animal validation can potentially reveal the underlying mechanism of biofabricating transplantable liver microtissues, laying the foundation for liver repair and regeneration of other organs.

体外构建肝组织/器官是解决急性肝衰竭和其他晚期肝病最有潜力的研究方案。本项目拟在体外调控三维（3D）动态仿生微环境从而构建具有成熟稳定肝功能并可体内移植的“微肝组织”，揭示微环境对微肝组织生成的作用机制。首先，调控生物化学因子将诱导性多能干细胞体外诱导生成肝实质细胞（iPS-Heps）和血管内皮细胞（iPS-Ends）。其次，采用水凝胶支架和微流控芯片制备技术构建仿生肝小叶的六边形载细胞支架；调控支架材料的刚度和孔隙结构以及微流控生物反应器中的生物化学因子和流体流速以构建适宜iPS-Heps和iPS-Ends共培养的3D动态仿生微环境，从而生成具有成熟稳定肝功能且可移植的微肝组织；最后，将具有成熟稳定肝功能的微肝组织移植到急性肝衰竭的小鼠体内。本项目将系统揭示细胞培养微环境诱导生成微肝组织的作用机制，研究微肝组织生成过程中的细胞学规律，同时为其他重要组织器官的修复和再生奠定基础。

体外构建肝组织/器官是解决急性肝衰竭和其他晚期肝病最有潜力的研究方案。本项目在体外调控三维（3D）动态仿生微环境从而构建可体内移植的血管化“微肝组织”，并用于修复小鼠急性肝衰竭。首先，采用生物化学因子分段诱导方法诱导iPSCs成功向肝细胞分化。其次，通过细胞共培养技术并结合光投影Z轴3D打印技术和生物反应器技术在体外成功构建血管化的微肝组织。最后，体内实验证明了血管化的微肝组织可以修复和治疗小鼠急性肝衰竭。这一研究为体外微肝组织构建提供了新方法，为肝脏的修复和再生提供了新思路，也为其他重要器官的重塑奠定了基础。