Development of a new hybrid-type aftificial liver support system using packed-bed type reactor

为了利用培养的肝细胞开发混合型人工肝支持系统(ALSS)，进行了以下尝试。混合型ALSS性能的动力学分析为获得优化设计和改进混合型ALSS的可靠标准，基于培养肝细胞的隔室传质模型和反应动力学，对ALSS的性能进行了理论和实验评价。用中空纤维型组件、转盘型组件和填充床型组件进行的体外和体外灌流实验结果表明，实验获得的组件在肝细胞活力和一些代谢功能方面的性能与理论估计的性能有很好的相关性。与全尺寸装置设计问题相关的模拟研究表明，ALSS的性能不可避免地受到肝细胞…的生存能力的限制更多的TES。结论：1.通过改善肝细胞培养条件，可以显著改善ALSS的性能。利用多孔树脂在填充床反应器中长期连续培养肝细胞我们研究了以网状聚乙烯醇缩甲醛(PVF)树脂为支撑材料的新型填充床式反应器中培养的肝细胞的长期代谢功能。使用填充床反应器(直径20 mm)进行长期(长达1周)的灌流培养实验。将500个PVF树脂块(平均孔径250微米，2*2*2 mm)负载，并与常规单层培养实验对照，在无血清或有血清的培养液中进行。以反应动力学分析为指标，定量评价培养肝细胞的氨代谢、尿素合成和白蛋白分泌活性。当无血清培养液用于灌流培养时，随着培养时间的延长，肝细胞功能显著衰退，不到第1天时的10%。这种活性丧失在灌流培养中比在单层培养中更为明显。相反，当使用含血清的培养液时，在灌流和单层培养实验中，即使在培养结束时，第一天获得的这些活性大约有50%保持不变。结论：PVF树脂填充床反应器可实现肝细胞的高密度培养，并能较长时间地维持固定化肝细胞的代谢功能，最长可达1周。因此，这种类型的反应器被认为是在克服混合型人工肝支持系统开发中涉及的困难方面的突破。较少

In order to develop a hybrid-type artificial liver support system (ALSS) using cultured hepatocytes, following attempts were carried out.1. Kinetic analyzes of the performance of a hybrid-type ALSSTo obtain sound criteria for the optimal design and improvement of the hybrid-type ALSS using isolated hepatocytes, theoretical and experimental evaluations of the performance of the ALSS were made based on the compartmental mass transfer model and reaction kinetics of the cultured hepatocytes. Results of in vitro and ex vivo perfusion experiments using a hollow-fiber type module, a rotating-disk type module or packed-bed type module indicated that experimentally obtained performance of the module in terms of hepatocyte viability and a few metabolic functions were well correlated with the theoretically estimated performances. Simulation studies related to the design problems of a full-scale device revealed that the performance of the ALSS is inevitably limited by the viability of the hepatocy … More tes. It was suggested that a considerable improvement in the performance of ALSS is to be attained by improving hepatocyte culture conditions.2. Long-term continuous culture of hepatocytes in a packed-bed reactor utilizing porous resinWe investigated the long-term metabolic function of hepatocytes incubated in a newly developed packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250mum, 2*2*2mm), together with conventional monolayr culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism, urea synthesis and albumin secretion activities were evaluated quantitatively based on reaction kinetic analyzes as indices of metabolic functions of the cultured hepatocytes. When serum-free medium was used in the perfusion cultures, hepatocytes functions showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayr cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayr culture experiments. It was concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain metabolic functions of the immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming difficulties involved in the development of a hybrid type artificial liver support system. Less