旋转生物反应器内微载体共培养CL-1肝细胞与人肝星形细胞

Evaluation of co-cultured CL-1 hepatocytes and hepatic stellate cells in rotatory cell culture system

目的评估cL-1肝细胞跟人肝星形细胞（HSC）的微载体微重力共培养来提高CL-1肝细胞的活力与功能的可行性。方法实验分为两组：CL-1肝细胞微载体微重力单培养组和CL-1肝细胞、HSC微载体微重力共培养组。通过倒置显微镜观察、细胞计数、MTT染色及扫描电子显微镜比较两组肝细胞的形态、细胞活力差异，并通过、培养上清中ALT、白蛋白浓度等功能指标测定，比较两组肝细胞活力和功能差异。结果共培养组较单培养组细胞生长迅速，细胞从24h贴壁后开始增长，到第5天进入高峰。共培养组1～7d的肝细胞密度明显高于单培养组的细胞密度（P〈0．05）。两组肝细胞ALT、白蛋白从第1天开始不断分泌增加，第5天达到峰值，第6、7天有所下降。ALT功能比较，共培养组明显低于单培养组（P〈0．05）。白蛋白功能比较，共培养组明显高于单培养组（P〈O．05）。倒置显微镜、扫描电镜及MTT均提示共培养组肝细胞形态、活力优于单培养组。结论人肝细胞、HSC微载体微重力共培养研究有利于维持及增强旋转生物反应器的肝细胞活力及功能，对人工肝的培养模式发展具有重要意义。

Objective To explore whether the function of the CL-1 hepatocytes, co-cultured with human hepatic stellate cells （HSC） on microearriers was better than that cultured without HSC. Methods CL-1 hepatocytes were divided into 2 groups. The co-culture group was cultured with HSC in DMEM culture medium containing 10% fetal bovine serum, and HSC were not added in single-culture group. The cytomorphology was observed by inverted microscope and scanning electron microscopy. The effects of different culture method on the proliferation in vitro were analyzed by MTT assay. The function of hepatocytes was evaluated through measuring the concentration of ALT and albumin. Results The inverted microscope and the MTT staining results showed that the quantity and viability of the human hepatocyte （C3A） in bidirectional bioreactor group were much better than the RCMW group. The growth curve results showed that the density of the human hepatocyte （C3A） was firstly increased and then decreased in both groups, and the peak of the curve appeared in day 5. The density of human liver cells in the second generation of bi-directional rotation and perfusion microgravity bioreactor group was significantly higher than the RCMW group from day1 to day 7 （P〈0.05）. The functional results showed that the albumin and urea concentration, which reached the peak on day 5, also gone up firstly and then gradually gone down in both teams. And the albumin and urea concentration in bidirectional bioreactor group was significantly higher than RCMW group from day 1 to day7 （P〈0.01）. Besides, the concentration of ALT and AST in bidirectional bioreactor group was significantly lower than RCMW group from day 1 to day 7 （P〈0.05）. Conclusions This study shows that this new culture method is advantageous in enhancing cell viability and function. It indicates that co-culturing hepatocytes with HSC has a good application prospect in the development of artificial liver technology.