Development of A Hybrid-type Artificial Liver Utilizing Isolated Hepatocytes

从工程学角度对异种肝细胞混合型人工肝支持系统（ALSS）的性能进行了理论和实验评价，并从肝细胞活力和代谢功能（以氨和己酸等几种溶质的代谢功能为代表）两个方面对肝细胞培养条件进行了优化，通过使用大鼠的分离肝细胞的分批培养实验测定。体外灌流实验的结果与基于三组分传质模型的理论估计性能有很好的相关性。仿真研究表明，ALSS的性能不可避免地受到肝细胞活力的限制。因此，ALSS的性能的改善也打算通过采用条件培养基，也通过捕获肝细胞在钙alginate水凝胶。通过对培养条件的这些改进，获得了与悬浮培养中相同水平的代谢活性和较低的死亡速率常数<（k_v）>。将肝细胞包埋在珠状或膜状海藻酸钙凝胶中，<k_v>其代谢速率常数较悬浮培养降低1 ~ 2个数量级，而代谢反应速率常数基本保持不变。利用该组件对肝衰竭家兔进行体外灌流实验，结果表明该组件具有良好的清除家兔血中铵离子的代谢性能。因此，本研究所开发的转盘式人工肝可望成为未来临床应用的有力候选者。

Theoretical and experimental evaluations and the improvement of the performance of a hybrid-type artificial liver support system (ALSS) utilizing heterogeneous isolated hepatocytes were made from the engineering points of view.Optimum conditions of the hepatocyte culture in terms of hepatocyte viability and metabolic functions, represented by metabolizing functions of a few solutes such as ammonia and caproic acid, were determined by batch culture experiments using isolated hepatocytes of the rats. Results of in vitro perfusion experiments were well correlated with the theoretically estimated performances based on the three compatments mass transfer model. Simulation studies demonstrates that the performance of ALSS is inevitably limited by the hepatocyte viability. Thus, the improvement of the performance of ALSS was also intended by employing the conditioned medium, and also by entrapping hepatocytes in calcium alginage hydrogel. With these modifications of culture conditions, the lower death rate constants <(k_v)> and about the same level of metabolic activity as in the suspension culture were obtained. In particular, entrapping of hepatocytes in bead-shaped or film-shaped calcium alginate gel resulted in a remarkable decrease in <k_v> by one to two order of magnitude smaller than that of suspension culture, while metabolic reaction rate constants remained almost unchanged.A new ALSS module was constructed by use of 30 discs made of stainless steel mesh, which was coated by a thin film of hepatocytes containing hydrogel, 6 cm in diameter and 1 cm in annular width and mounted on a horizontally rotating axis. Extracorporeal purfusion experiments by means of this module on hepatic failure rabbits showed satisfactory metabolic performance of the module to eliminate ammonium in the rabbit blood. Thus, the ratating disc type artificial liver developed in the present project is expectable as a powerful candidate of future clinical use.