Use of Discarded Organs for Preparation of Liver Grafts

描述（由申请人提供）：终末期器官衰竭的治疗受到供体器官严重短缺的限制，器官等待名单目前有10万份申请，并以每年5%的速度增加。肝脏的问题也不例外，这项研究的重点是肝脏——在美国，约有4000人因缺乏可移植器官而死亡，而缺乏捐献器官被认为是一项重大的健康危机。此外，由于移植往往可以解决许多与衰老有关的疾病，估计隐性需求远远超过目前的水平。这种情况是组织工程兴起的主要推动力，器官衰竭治疗的市场估计约为800亿美元。然而，20多年来旨在从头开始构建组织的工作尚未成功地创造出可用于临床植入的大规模组织，以解决器官替代疗法中的空白。此外，尽管在干细胞研究方面做出了巨大的努力，包括我们的研究小组，但缺乏用于细胞治疗的初级成人肝细胞的可靠细胞来源的问题仍然存在，而且在不久的将来不太可能得到解决。有趣的是，有许多潜在的器官捐献者因为器官受损而没有被考虑进行移植。例如，在心脏死亡后到达医院的事故受害者由于过度的缺血性损伤而不是合格的捐赠者；即使是轻微的缺血性损伤（>30min热缺血或>16h冷缺血）也会导致长期并发症，6个月时移植物存活率显著降低。据估计，在美国，心脏死亡（DCD）后的潜在捐赠者人数每年超过20万，其中约有6000人被认为只是轻微受损。我们的长期目标是设计用于治疗或治疗相关肝脏疾病的可移植肝移植。该研究的目的是开发人源化重组肝移植作为一种可行的体外肝脏模型。在该奖项的指导阶段（K99），将开发两个基本工具来实现这一目标：1)肝脏灌注系统，这将使边缘供体肝脏恢复健康的肝细胞。这项技术有望建立一个目前尚未开发的成人肝细胞来源，以满足对人类细胞的需求，直到干细胞接近成熟并安全有效地用于临床。2)全器官灌注-脱细胞和再细胞化方法。目的是开发一种新的组织工程支架，支持细胞附着和功能，并可血管化。在该奖项的独立阶段（R00），主要目标是将K99阶段开发的方法扩展到大型动物模型并最终应用于人体器官。本项目提出的工作预计将：(1)建立边缘肝脏作为原代肝细胞的可靠来源，(2)建立脱细胞肝切片作为新的3D细胞培养平台，研究ECM的作用，(3)开发人源化大鼠肝移植作为药物研究的三维肝脏模型，以及(4)导致开发用于治疗肝脏疾病的重组肝移植。虽然这项工作利用肝脏作为模型器官，但这项工作的结果也将产生积极的影响，为未来复杂的器官工程技术奠定基础，这些技术包括多种不同的细胞类型，并可以转化为其他器官（如胰腺为胰腺β细胞移植创建血管化补丁），并可能最终导致体外整个器官的发展。

DESCRIPTION (provided by applicant): Treatment for end-stage organ failure is restricted by the critical shortage of donor organs with the organ waiting list currently at 100,000 requests and it is increasing by 5% every year. The problem is not different for liver, which this study focuses on - about 4,000 people die in the US due to lack of a transplantable organ, and the lack of donor organs is considered a major health crisis. In addition, since transplantation can often be the solution to many aging related diseases, the hidden demand is estimated to be far beyond the current levels. This situation has been a major driving force behind the rise of tissue engineering, with the market for organ failure treatments estimated at about $80 billion. However, over two decades of work aimed at building tissues from the ground up has not succeeded in creating large-scale tissues that can be clinically implanted to address the void in organ replacement therapies. Further, despite intense efforts on the stem cell front, including those from our group, the lack of a reliable cell source for primary adult hepatocytes for use in cell therapies persists and is unlikely to be resolved in the near future. Interestingly enough, there are many potential organ donors that are not considered for transplantation because the organs are damaged. For example, accident victims who arrive at the hospital after cardiac death are not eligible donors because of excessive ischemic damage; even a slight ischemic damage (>30min warm ischemia or >16hrs cold ischemia) is known to lead to complications in the long term with significantly reduced graft survival at 6 months. By some estimates, potential number of Donors after Cardiac Death (DCD) is over 200,000 per year in the US, and about 6,000 are considered to be only marginally damaged. Our long-term goal is to engineer transplantable liver grafts for curing or treating relevant liver diseases. The objective of the proposed study is to develop humanized reengineered liver grafts as a viable in vitro liver model. During the mentored phase (K99) of the award, two essential tools will be developed to reach this goal: 1) a liver perfusion system, which will enable recovery of healthy hepatocytes from marginal donor livers. This technology is expected to lead to establishment of a currently untapped source of adult human hepatocytes that will fill the need for human cells until stem cell approaches mature and become safe and efficient for clinical use. 2) a whole organ perfusion- decellularization and recellularization methodology. The objective here is to develop a novel scaffold for tissue engineering, which supports cell attachment and function and is vascularizable. During the independent phase (R00) of the award, the primary goal is the scaling of the methods developed in K99 phase to large animal models and ultimately human organs. The work proposed in this project is expected to i) establish marginal livers as a reliable source of primary hepatocytes, ii) establish decellularized liver slices as novel 3D cell culture platform to study the role of ECM, iii) develop humanized rat liver grafts as a three dimensional liver model for pharmaceutical studies, and iv) lead to the development of reengineered liver grafts to treat liver diseases. While this work utilizes liver as the model organ, the results of this work will also have a positive impact by establishing the basis of future sophisticated organ engineering techniques that incorporate multiple different cell types and can be translated to other organs (such as pancreas to create vascularized patches for pancreatic beta-cell transplantation), and may ultimately lead to development of entire organs in vitro.