Use of Discarded Organs for Preparation of Liver Grafts

项目总结/摘要 终末期器官衰竭的治疗受到器官供体严重短缺的限制 等候名单目前有10万个申请，每年以5%的速度增长。问题是没有不同的 肝脏，这项研究的重点-大约4,000人死于美国由于缺乏可移植的器官， 缺乏捐赠器官被认为是一个重大的健康危机。此外，由于移植往往是 由于许多与衰老有关的疾病的解决方案，估计隐藏的需求远远超过目前的水平。 这种情况一直是组织工程兴起的主要推动力， 失败治疗估计约为800亿美元。然而，二十多年来旨在构建组织的工作 从根本上说，还没有成功地创造出可以在临床上植入的大规模组织， 填补器官替代疗法的空白此外，尽管在干细胞方面付出了巨大的努力， 包括来自我们组的那些，缺乏用于细胞培养的原代成体肝细胞的可靠细胞来源， 治疗持续存在，不太可能在不久的将来得到解决。有趣的是， 由于器官受损而不考虑移植的潜在器官捐献者。为 例如，心脏病死亡后到达医院的事故受害者不符合资格的捐赠者，因为 过度缺血性损伤;甚至轻微缺血性损伤（> 30分钟热缺血或> 16小时冷缺血） 已知会导致长期并发症，6个月时移植物存活率显著降低。一些 据估计，在美国，心脏死亡后（DCD）的潜在供体数量每年超过20万， 约有6 000个被认为只是轻微受损。我们的长期目标是设计出可移植的 用于治疗或治疗相关肝脏疾病的肝脏移植物。拟议研究的目的是发展 人源化再工程肝移植物作为可行的体外肝模型。在辅导阶段（K99）， 为了实现这一目标，将开发两种基本工具：1）肝脏灌注系统， 从边缘供体肝脏中回收健康肝细胞。这项技术预计将导致 建立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 直到干细胞方法成熟并变得安全有效用于临床。2)整个器官灌注 脱细胞化和再细胞化方法。目的是开发一种新的组织支架， 工程化，其支持细胞附着和功能并且是可血管化的。在独立阶段 (R00)该奖项的主要目标是将K99阶段开发的方法扩展到大型动物 模型和最终的人体器官本项目中提出的工作预计i）建立边际 肝脏作为原代肝细胞的可靠来源，ii）建立脱细胞肝切片作为新的3D细胞 培养平台以研究ECM的作用，iii）将人源化大鼠肝移植物开发为三维肝脏 用于药物研究的模型，以及iv）导致开发重组肝移植物以治疗肝脏 疾病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具有积极的意义。 通过建立未来复杂的器官工程技术的基础， 不同的细胞类型，并可以转移到其他器官（如胰腺，以创建血管化的补丁， 胰腺细胞移植），并可能最终导致整个器官的体外发育。

PROJECT SUMMARY/ABSTRACT 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 ¿-cell transplantation), and may ultimately lead to development of entire organs in vitro.