Development of Hybrid Artificial Liver with Isolated Hepatocytes in Extracorporeal Circulation

为了尝试使用分离的肝细胞开发混合型人工肝（HAL），我们将肝细胞包裹在胶原凝胶中制备中空纤维模块，并在肝衰竭模型中评估HAL装置。并研究了胶原凝胶包埋肝细胞的低温保存（4c），以保存HAL装置。在大鼠实验中，采用体外血液灌流法对含有4*10^7个大鼠肝细胞的模块进行评价。在缺血性肝衰竭模型中，该装置显示氨去除和糖异生。药物（半乳糖胺）诱导的急性肝衰竭大鼠体外血液灌流中总胆汁酸和GOT均有改善。该装置展示了对宿主肝脏功能的支持。在家兔实验中，对含有3.5*10^8个兔肝细胞的模块进行评价。在第0天进行灌注培养，结果显示良好地维持了白蛋白分泌、尿生成和氨去除的分化肝功能。灌注液中出现与兔血清中相同分子量的蛋白。通过对无肝兔进行一个或两个模块的体外血液灌流，对该装置进行了评价。在单一HAL组中，血压明显维持。抑制血清氨的升高，显著延长存活时间。双HAL组乳酸、凝血酶原时间和Fisher比值显著提高。这些研究为我们的HAL系统包括解毒、合成和代谢功能提供了证据，并可能在未来的临床应用中有所帮助。我们研究了胶原凝胶包裹的肝细胞在4 - <s:1>大学威斯康星分校溶液中的保存。保存的肝细胞在相位差和扫描电镜下呈圆形形态。即使保存了14天，它们仍然保持了足够的功能。

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.