High Yield Human Hepatocyte Isolation

描述(由申请人提供)：在美国，终末期肝病夺走了大约30,000人的生命。许多患者病情严重，无法耐受肝移植，即使移植 有迹象表明，可行的捐赠器官严重短缺，只有28%的等待名单上的患者接受了移植。器官移植的新替代品，如生物人工肝(BAL)装置、细胞移植和组织工程肝替代品的开发和翻译研究，由于缺乏可靠的人类肝细胞来源而受到严重制约。然而，大量未使用的人体细胞存在于废弃的供体肝脏中，如心脏死亡后的供体(DCD)，由于热缺血损伤而无法移植，这大大降低了移植的成功率。最近的研究，包括我们小组的研究表明，缺血和其他边缘肝脏可以通过体外机器灌流(MP)进行修复。据估计，这种轻微受损的肝脏可以挽救并修复以进行移植的数量约为6,000；更多的肝脏可能损伤得太严重而无法移植，但仍可作为细胞来源。人类细胞的这一巨大来源仍未被探索。我们的长期目标是开发机器灌流，作为一种治疗目前未使用的供体肝脏的方法，并增加供体肝脏的可用性 移植、基于细胞的治疗和研究。这项STTR验证研究的目的是建立因过度缺血而不符合移植条件的人肝，作为高质量原代肝细胞的可靠来源。这里要检验的中心假设是，机器灌流前处理增加了从缺血的人肝脏中恢复肝细胞的产量。提高不合格的人供体肝脏的肝细胞活性产量是一项创新的努力，因为它利用了我们小组开发的新的机器灌流方法，在大鼠模型中将肝细胞产量提高了25倍。确定可靠的人类肝细胞来源将极大地改变药物发现和测试的实践，允许在早期和所有水平的药物测试中使用人类肝细胞，减少对毒理学动物模型的依赖，从而降低药物开发成本。以低成本获得高质量的人类肝细胞也将消除实现基于细胞的肝功能衰竭治疗的一个主要瓶颈。

DESCRIPTION (provided by applicant): End-stage liver disease claims about 30,000 lives in the US. Many patients become too ill to tolerate liver transplantation, and even if transplantation were indicated, there is a severe shortage of viable donor organs and only 28% of the wait-listed patients receive transplants. Development and translational studies with novel alternatives to organ transplantation, such as Bioartificial Liver (BAL) devices, cell transplantation and tissu engineered liver substitutes are severely bottlenecked by the lack of a reliable source of viable human hepatocytes. However, a vast pool of unused human cells lies in the discarded donor livers, such as Donors after Cardiac Death (DCD), which are not transplanted due to warm ischemic injury, which significantly reduces transplant success. Recent studies, including those from our group, demonstrate that ischemic and other marginal livers can be reconditioned through extracorporeal Machine Perfusion (MP). The estimated number of such marginally damaged livers that could be rescued and reconditioned for transplantation is ~ 6,000; many more are likely to be too damaged for transplantation, but still viable as a cell source. This immense source of human cells remains unexplored. Our long-term goal is to develop machine perfusion as a way to treat currently unused donor livers and increase donor liver availability for transplantation, cell-based therapeutics and research. The objective of this STTR validation study is to establish human livers disqualified for transplantation because of excessive ischemia, as a reliable source of high quality primary hepatocytes. The central hypothesis to be tested here is that machine perfusion pre-treatment increases the yield of hepatocyte recovery from ischemic human livers. Enhancing the viable yield of hepatocytes from disqualified human donor livers is an innovative endeavor as it capitalizes on the new machine perfusion method developed by our group and increases hepatocyte yield by 25 fold in the rat model. Identifying a reliable human hepatocyte source would dramatically alter the practice in drug discovery and testing by allowing use of human hepatocytes early and at all levels of pharmaceutical testing, reduce reliance on animal models for toxicology, and therefore lead to reduced cost for drug development. Availability of high quality human hepatocytes for a low cost would also eliminate a major bottleneck in realization of cell-based therapies for liver failure.