基于iPSCs生物人工肝反应器的构建与评估

生物人工肝可能是肝衰竭治疗中极具潜力的治疗手段之一，但原代肝细胞扩增困难、猪肝细胞和肝癌细胞的安全性争议和体外培养失去肝脏微环境等原因使得生物反应器制备出现瓶颈。诱导性多能干细胞（iPSCs）具备大量增殖和肝向分化特性；肝细胞与非实质细胞的共培养及适宜剪切力能模拟肝脏微环境，能够进一步促进肝细胞成熟及功能表达。本项目将iPSCs与模拟肝脏微环境相结合，诱导iPSCs肝向分化，通过共培养+适宜流体剪切力模拟肝脏微环境中细胞-基质-微血流的相互作用，研究共培养和剪切力在肝细胞分化成熟、细胞团微结构形成中的作用，探讨钙离子转运和肝细胞生长因子产生在细胞团应对机械力中的具体作用；建立共培养+适宜流体剪切力的最佳方案，设计制备多层板培养支架，构建新型生物反应器并进行体内功能验证。本项目结合基础研究与应用转化，探讨共培养和剪切力对于干细胞分化的具体影响，最终构建新型生物反应器，为肝衰竭治疗提供新选择。

Bioartificial-liver is a novel and potential method to solve liver failure. However, due to the difficulties of primary hepatocyte expansion, the controversial safety of porcine hepatocytes and hepatoma cells, the low function of cells, and the loss of microenvironment characteristics of liver in vitro, the preparation of bioreactor was limited. Induced pluripotent stem cells (iPSCs) have potential for proliferation and differentiation into hepatocytes with no ethical dispute about stem cells. co-culture of hepatocytes and nonparenchymal cells and fluid shear can further promote the maturation and functional expression of hepatocytes. In this project, iPSCs-Hep will be used as functional cell, and we will create a similar microenvironment to simulate the interaction among cell-matrix-blood flow in liver microenvironment through combining co-culture and fluid shear. the roles of co-culture and fluid shear in the differentiation and maturation of hepatocytes, the formation of tissue microstructure and the expression of function will be studied. The specific role of Ca2 + transport and HGF secretion in the response of cell clusters to mechanical forces will be explored. A new culture scheme of fusion of co-culture and fluid shear will be explored, then multi-layer plate culture stent and a new type bioreactor will be constructed on this basis. In this project, in combined with basic research and application transformation, we will explore the specific effects of co-culture and fluid shear on stem cell differentiation, ultimately build a new type bioreactor for bioartificial-liver, and provide a new choice for treatment of liver failure.