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Development of bioartificial liver using human hepatocytes

利用人肝细胞开发生物人工肝

基本信息

批准号：
09557105
负责人：
IKAI Iwao
金额：
$ 8.13万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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.

1.猪肝细胞和人肝细胞的长期培养及其形态学和功能学评价为了建立以人肝细胞为原料的生物人工肝，我们对一种新的人肝细胞长期培养方法进行了研究。它们的存活率在分离后约为90%，在冷冻保存后约为70-80%。这些猪肝细胞能够在改良的培养基中培养，其中成纤维细胞不增殖3个月，保持高细胞色素p450活性。2个月后，这些细胞在形态学上被证实为肝细胞。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，并观察到少数细胞在早期增殖.生物人工肝生物反应器的研制采用临床上用于血液透析的中空纤维滤芯作为生物人工滤芯。药筒中的中空纤维由标称截止分子量为70 kDa的三乙酸纤维素制成。将分离的猪肝细胞接种到中空纤维的内部空间中。通过加载化学物质如氨、利多卡因和半乳糖来评价接种猪肝细胞的生物反应器的功能。这些化学物质在加载至少10天后被消除，并且中空纤维中的肝细胞形成具有重建的紧密连接和小梁样结构的组织良好的聚集体。