Development of Hybrid Artificial Liver with Isolated Hepatocytes in Extracorporeal Circulation

为了利用分离的肝细胞研制混合型人工肝(HAL)，我们用胶原凝胶包裹肝细胞制备了中空纤维组件，并在肝功能衰竭模型中对HAL装置进行了评价。并对胶原胶包裹的肝细胞低温保存(4ﾟC)进行了研究。在大鼠实验中，对含有4×10^7个大鼠肝细胞的模块进行了体外血液灌流。在缺血性肝功能衰竭模型中，该装置显示了氨的清除和糖异生。药物(氨基半乳糖)诱导的急性肝功能衰竭大鼠体外血液灌流中总胆汁酸和谷草转氨酶均有改善。在兔实验中，对含有3.5×10^8个兔肝细胞的组件进行了评估。灌流培养表明，在第0天，白蛋白分泌、尿液生成和氨排出等分化的肝脏功能保持良好。灌流液中出现一种与兔血清分子量相同的蛋白质。通过使用一个或两个模块的体外血液灌流，在无肝兔体内评价该装置为HAL。在单一HAL组中，血压显著维持。血氨升高受到抑制，存活时间明显延长。在双HAL组中，乳酸、凝血酶原时间和Fisher比率明显改善，这些研究为我们的HAL系统具有解毒、合成和代谢功能提供了证据，并可能在未来的临床上应用。我们研究了胶原凝胶包裹的肝细胞在4ﾟC威斯康星大学溶液中的保存。本系统保存的肝细胞在相差显微镜和扫描电子显微镜下呈圆形。即使在保存14天后，它们仍保持着足够的功能能力。

In an attempt to develop a hybrid artificial liver (HAL) using isolated hepatocytes, we prepared hollow fiber modules with hepatocytes entrapped in collagen gel and evaluated the HAL devices in hepatic failure models. And we studied the hypothermic preservation (4ﾟC) of hepatocytes entrapped in collagen gel, for the purpose of storage of the HAL devices.In rats experiments, the modules contained 4*10^7 rat hepatocytes were evaluated by extracorporeal hemoperfusion. In ischemic hepatic failure models, the device revealed ammonia removal and gluconeogenesis. Drug (galactosamin) induced acute hepatic failure rats showed the improvement of total bile acid and GOT in extracorporeal hemoperfusion. The devices demonstrated the supporting failing functions on host livers.In rabbits experiments, the modules contained 3.5*10^8 rabbit hepatocytes were evaluated. Perfusion culture was performed and demonstrated good maintenance of differentiated liver functions of albumin secretion, ureogenesis and ammonia removal at Day 0. Perfusion solution showed appearance of a protein with same molecular weight as in rabbit serum. The devices were evaluated as a HAL by extracorporeal hemoperfusion using one or two modules in anhepatic rabbits. In the single HAL groups, blood pressure were significantly maintained. Increase in serum ammonia was inhibited and survival time were significantly prolonged. In the double HAL groups, lactic acid, prothrombin time and Fisher ratio were improved significantly.These studies provided the evidence that our HAL system included detoxicating, synthetic and metabolic functions, and it may be clinically useful in the future.We studied the preservation of hepatocytes entrapped in collagen gel in 4ﾟC University of Wisconsin solution. Preserved hepatocytes in our system showed round-shaped morphology in phase-difference and scanning electron microscopy. They maintained adequate functional capability even after 14 days of preservation.