Development of bioartificial liver using human hepatocytes

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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.