Clinical application of bioartifical liver support and heopatocyte transplantation using human hepatocytes

肝移植提高了危及生命的肝病患者的生存率和生活质量。然而，这种疗法与几个问题有关，包括显著的发病率、死亡率和肝脏供体短缺。因此，临床上需要使用生物人工肝和分离肝细胞。不幸的是，可用于人肝细胞分离的供体肝脏数量有限。其他动物种属已被建议作为这些治疗的肝细胞的潜在来源。然而，跨物种使用的免疫和生理障碍尚未克服。本研究的目的是建立一种高效的人肝细胞库的分离和冻存方法。此外，HVJ-脂质体的效率为基因转移到猪hepatocystein.Results：人肝细胞收获16名志愿者与转移性肝肿瘤或血管瘤使用修改的两步胶原酶灌注技术。新鲜分离肝细胞的产量平均超过8.5 × 10^6/肝克，存活率为80%。冻存30天的解冻肝细胞在UW（University of威斯康星州）人新鲜冰冻血浆（FFP）液中的平均存活率为62%，平板接种率为40%。UW液和FFP的使用显著提高了冻存人肝细胞的存活率和平板接种效率，目前我们保存的人肝细胞为3.0 × 10 ^<10>。因此，我们可以治疗三个致命的肝衰竭患者。但目前尚未见肝细胞介导的转基因治疗病例，HVJ-脂质体载体是一种安全可行的猪肝细胞介导的转基因方法。该方法可用于肝细胞基因治疗的临床试验。

Liver transplantation improves the survival rate and quality of life for patients with life-threatening liver diseases. This therapy, however, is associated with several problems including significant morbidity, mortality and the shortage of liver donors. Therefore, the demand for clinical use of bioartificial liver and isolated hepatocytes. Unfortunately, the number of donor livers available for human hepatocyte isolation is limited. Other animal species have been suggested as a potential source of hepatocytes for these therapies. However, immunologic and physiologic barriers to cross-species use have yet to be overcome. In addition, there is another concern about zoonosis so that human hepatocytes shoud be used for these therapies.The purpose of this study is to establish an efficient isolation and cryopreservation method for the human hepatocyte bank. In addition, efficiency of HVJ-liposome for gene transfer into porcine hepatocytes was investigated.Results :Human hepatocytes were harvested from 16 volunteers with metastatic liver tumors or hemangiomas using a modification of the two step-collagenase perfusion technique. The yield of freshly isolated hepatocytes averaged more than 8.5x10^6/liver gr.with 80% viability. The average viability of thawed hepatocytes that had been cryopreserved for 30days was 62% and plating efficiency was 40% in the solution of UW (University of Wisconsin) with human fresh frozen plasma (FFP). The use of UW solution and FFP significantly improved the viability and plating efficiency of cryopreserved human hepatocytes.Now, we preserve 3.0x10^<10> human hepatocytes. Therefore, we can treat three patients with fatal liver failure. However, there have been no patients for the hepatocyte-based therapy during this study.HVJ-liposome vector was safe and feasible modality for hepatocyte-directed gene transfer in pigs. It might be suitable for clinical hepatocyte-gene therapy trials.