Development of bioartificial liver support utilizing hepatocyte spheroids and its evaluation on pigs with liver failure.

研制了一种利用肝细胞多细胞球体的生物人工肝支持体。分离的大鼠或猪肝细胞在化学成分确定的培养基和蛋白聚糖及其类似物的培养基质中进行原代培养，使其形成球状体。为了大规模生产球状体猪肝细胞，在用硫酸软骨素和磷脂酰乙醇胺化学合成的新蛋白聚糖预包被的多层板中培养。以p-450酶活性、白蛋白生成和尿素生成为指标评价球状体的功能水平。生物人工肝支持系统由一个装有肝细胞球体的生物反应器组成。闭合回路配备了调节器单元，可自动调节循环介质中溶解氧和pH值的水平。使用用于血浆交换的中空纤维筒或喷动床室作为生物反应器室。在D-氨基半乳糖诱导的急性肝衰竭猪模型上评价了生物人工肝支持系统的性能。生物人工肝支持系统治疗组血氨水平均明显降低。但存活率没有显著提高，但部分猪的存活时间有所延长。这些结果是用相当于体内肝细胞体积约10%的肝细胞球体获得的。

A bio-artificial liver support utilizing hepatocyte multicellular spheroids (spheroid) were developed. Isolated rat or porcine hepatocytes were allowd to form spheroids in the primary culture using chemically defined medium and culture substrates of proteoglycans and their analogs. For the large scale production of spheroid porcine hepatocytes were cultured in the multilayr-plates pre-coated with neo-proteoglycan that was chemically synthesized with chondroitin sulfate and phosphatidyl ethanolamine. Functional level of the spheroids were evaluated with the activities of p-450s, albumin production and urea production. A bio-artificial liver support system was consisted with a closed circuit to which a bioreactor chambercontaining hapatocyte spheroids was inserted. The closed circuit was equipped with a regulator unit that automatically regulated the levels of dissolved oxygen and pH in the circulating medium. A hollow fiber cartridge for plasma exchange or a spouted bed chamber were used as the bioreactor chamber. The performance of the bio-artificial liver support system was evaluated on pigs with acute liver failure experimentally induced by the administration of D-galactosamine. The reductio of serum ammonia level was observed in all of the pigs treated with bio-artificial liver support system. However no significant improvement of the survival rate was seen, though the elongation of survival time was in some pigs. These result obtained with hepatocyte spheroid equivalent to about 10% of liver cell volume in vivo.