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、死亡率和肝供体短缺。因此，临床应用生物人工肝和分离肝细胞的需求越来越大。不幸的是，可用于人类肝细胞分离的供体肝脏数量有限。其他动物物种也被认为是这些治疗的潜在肝细胞来源。然而，跨物种使用的免疫和生理障碍尚未克服。此外，还有另一个关于人畜共患病的担忧，因此人类肝细胞应该用于这些治疗。本研究旨在建立一种高效的人肝细胞库分离和低温保存方法。此外，我们还研究了hvj脂质体在猪肝细胞中的基因转移效率。结果：使用改进的两步胶原酶灌注技术，从16名患有转移性肝肿瘤或血管瘤的志愿者身上采集了人肝细胞。新分离的肝细胞产量平均大于8.5x10^6/肝细胞，存活率为80%。解冻后的肝细胞在人新鲜冷冻血浆（FFP） UW （University of Wisconsin）溶液中冷冻保存30天，平均存活率为62%，镀层效率为40%。UW溶液和FFP的使用显著提高了人肝细胞冷冻保存后的存活率和镀膜效率。现在，我们保存了3 × 10^<10>个人肝细胞。因此，我们可以治疗3例致命性肝衰竭患者。然而，在本研究中没有患者接受肝细胞为基础的治疗。hvj脂质体载体是一种安全可行的猪肝细胞定向基因转移载体。它可能适用于临床肝细胞基因治疗试验。

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.