Development of bioartificial liver using human hepatocytes

1. 猪肝细胞与人肝细胞的长期培养及其形态和功能评价为了研制人肝细胞生物人工肝，我们研究了一种长期培养人肝细胞的新方法。采用胶原酶消化法分离正常猪肝细胞。分离后存活率约为90%，低温保存后存活率约为70-80%。这些猪肝细胞可以在改性培养基中培养3个月，其中成纤维细胞不增殖，保持较高的细胞色素p450活性。用胶原酶消解法分离的人肝脏肝细胞用改性培养基培养。2个月后形态学证实为肝细胞。3个月后，这些肝细胞保留了肝细胞特异性功能，尽管细胞数量减少，并且在早期阶段观察到少数细胞增殖。生物人工肝生物反应器的研制利用临床已应用于血液透析的中空纤维滤筒作为生物人工滤筒。筒内中空纤维由三醋酸纤维素制成，标称截止分子量为70 kDa。将分离的猪肝细胞接种到中空纤维的内部空间。通过加载氨、利多卡因和半乳糖等化学物质来评价生物反应器接种的猪肝细胞的功能。这些化学物质在加载至少10天后被消除，空心纤维中的肝细胞形成了组织良好的聚集体，重新建立了紧密连接和小梁样结构。

1. Long-term culture of porcine and human hepatocytes and their morphological and functional evaluationTo develop a bioartificial liver using human hepatocytes, we evaluated a new culture method for long-term culture of human hepatocytes.Porcine hepatocytes were isolated from normal pigs with collagenase digestion method. Their viability was approximately 90 % after isolation and 70-80% after cryopreservation. These porcine hepatocytes were able to be cultured with modified culture medium in which fibroblasts were not proliferated for 3 months, maintaing high cytochrome p450 activity Human hepatocytes isolated from human liver by collagenase-dispase digestion method were cultured with modified culture medium. These cells were confirmed as hepatocytes morphologically 2 months later. These hepatocytes preserved hepatocyte-specific functions 3 months later, in spite of a decrease of cell number and a few cells were observed to proliferate in an early phase.2. Development of bioreactor for bioartificial liverA hollow fiber cartridge that has been clinically used for hemodialysis was used for a bioartificial cartridge. The hollow fiber in the cartridge was made of cellulose triacetate with nominal cut-off molecular weight of 70 kDa. The isolated porcine hepatocytes were inoculated into the inner space of the hollow fibers. The function of bioreactor inoculated porcine hepatocytes was evaluated by loading chemicals such as ammonia, lidocaine, and galactose. These chemicals were eliminated after loading for at least 10 days, and the hepatocyte in the hollow fibers formed well-organized aggregates with reestablished tight junction and trabecular like structure.