Development of A Hybrid-type Artificial Liver Utilizing Isolated Hepatocytes

从工程角度对异种分离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，并通过大鼠肝细胞分批培养实验，确定了肝细胞活力和代谢功能的最佳培养条件，以氨和己酸等几种溶质的代谢功能为代表。体外灌流实验结果与基于三组分传质模型的理论估算结果吻合较好。仿真研究表明，ALSS的性能不可避免地受到肝细胞活性的限制。因此，通过使用条件培养液和将肝细胞包埋在藻酸钙水凝胶中，也可以改善ALSS的性能。通过这些培养条件的改变，获得了较低的死亡速率常数lt；(Kv)和gt；，而代谢活性与悬浮培养时的代谢活性水平大致相同。特别是，将肝细胞包埋在珠状或膜状海藻酸钙凝胶中，可使肝细胞的代谢反应速率常数几乎保持不变，但比悬浮培养明显下降1~2个数量级。用直径6 cm、环形宽1 cm的含有水凝胶的肝细胞薄膜包裹30个圆盘构成ALSS模块。用该模块对肝功能衰竭兔进行体外灌流实验表明，该模块具有良好的代谢性能，可清除兔血中的铵。因此，本项目所开发的旋转盘型人工肝有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.