去细胞化全肝生物支架循环灌注培养条件下体外细胞再植

In vitro cell replantation of decellularized liver biological scaffold under circulating-perfusion culture conditions

背景:通过去细胞化技术制备全肝生物支架成为缓解供体短缺的新技术,优化肝脏组织的去细胞化流程成为新的课题。目的:通过去细胞化技术建立完整保留肝脏三维结构和脉管系统的全肝生物支架,利用自主设计的循环灌注培养装置实现生物支架体外细胞再植。方法:通过门静脉路径循环灌注去垢剂TritonX-100,十二烷基硫酸钠,并用磷酸盐缓冲液洗脱残留去垢剂。通过动态循环灌注培养装置进行支架与HepG2细胞的共培养,观察植入细胞在全肝生物支架内的功能表达。结果与结论:经去垢剂灌注后,支架呈现保持肝脏三维结构的透明结构,苏木精-伊红染色以及扫描电镜结果显示细胞成分被完全移除,Masson’sTrichrome染色可见大量胶原纤维,免疫组化结果证实纤维连接蛋白和层粘连蛋白保存完整。循环灌注培养下细胞白蛋白表达量及尿素合成量较平板培养明显提高。说明去细胞化肝脏生物支架可作为体外肝脏组织重建的基础材料,动态循环灌注方法可实现支架中细胞再植。

BACKGROUND： The liver biological scaffold made by decellularization technology is the new technology to alleviate the shortage of donor; the optimization of the process to liver tissue decellularization is the new topic. OBJECTIVE： To establish the liver biological scaffold preserving the liver three-dimensional structure and the intact vascular system by decellularization technology, then, to realize the in vitro cell replantation of liver biological by scaffold independent designed circulating-perfusion culture device. METHODS： The cannula in the portal veins were attached to the pump and was perfused sequentially with TritonX-100 and sodium dodecyl sulfate, and then was perfused with phosphate buffered saline to wash out the residual detergent. The liver biological scaffold was cultured with HepG2 cells through the circulating-perfusion culture device and the expression of the cells in the liver biological scaffold was observed. RESULTS AND CONCLUSION： After the perfusion of the detergent, the scaffold was retained the transparent structure of the liver three-dimensional structure. Hematoxylin-eosin staining and scanning electron microscope reveled that the cellular components were completely removed. Masson’s Trichrome staining showed that there was a large amount of collagen fibers; the immunohistochemical results confirmed that fibronectin and laminin preserved intactly. Expression of cell albumin and the volume of the urea synthesis in the circulating-perfusion culture were significantly increased compared with plate culture. It indicate that the decellularized liver biological scaffold can be used as the basis materials of in vitro liver tissue reconstruction, and the dynamic circulating-perfusion culture method can achieved the cell replantation in the scaffold.