Extended Storage of Tissues and Organs in Subzero Environments

摘要 目前在美国有大约10万名患者在器官移植等待名单上， 超过了可用器官的供应，并且每年继续增长~5%。最有前途的 解决方案，生物人工组织和器官构建以及供体器官再造方法， 最终受到生物保存技术的限制，如在实验室中制备的任何组织工程产品 将不得不存储一段时间直到使用。目前整个器官的黄金标准 保存是在冰上冷藏长达72小时，在此期间器官不断恶化。一 将组织储存时间延长到超过当前限制的上级生物保存方法还有待开发 开发这种方法将为组织和器官的保存提供一种关键的使能技术， 组织和器官运输以及组织和器官移植。 本研究的目的是延长肝组织的存活保存时间， 在过冷保存介质中冷冻（SZNF）储存。这项研究的核心假设依赖于 两种现象：1）3-O-甲基-葡萄糖（3 OMG）降低了可达到的稳定SZNF温度， 主要毒副作用，2）常温灌注复温可减少再灌注损伤。我们 根据我们初步的发现，我们提出了一个假设，即3 OMG是一种毒性最小的药物， 冷冻保护剂的肝细胞，并建立常温灌注可以显着逆转 局部缺血的损害作用。这项研究的基本原理是，如果可以实现过冷保存， 同时避免防冻剂毒性，那么器官的新陈代谢可以进一步减慢，从而减少 缺氧/缺血性损伤降至最低水平。 建立一个零下不冻结保存技术将是一个受欢迎的创新领域。 本文所述的工作将有助于开发这种过冷存储的使能技术， 建立评价肝脏和生物人工器官保存后活力的定量标准。 当我们关注肝脏时，我们希望这里建立的协议也将作为以下方面的基础： 其他组织工程产品的低温非冷冻保存，如人造器官替代品， 接种的支架构建体。

ABSTRACT There are currently ~100,000 patients on the organ transplant waiting list in the US, a number that far exceeds the supply of available organs, and that continues to grow ~5% each year. The most promising solutions, bioartificial tissue and organ construction and donor organ reengineering methodologies, are both ultimately limited by biopreservation technologies, as any tissue engineered products prepared in a laboratory will have to be stored for a period of time until utilization. The current gold standard for whole organ preservation is cold storage on ice for up to 72 hours, during which time the organ continuously deteriorates. A superior biopreservation method that extends the tissue storage time beyond current limitations is yet to be developed. Such a method would provide a crucial enabling technology for tissue and organ preservation, tissue and organ transport, and tissue and organ transplantation. The objective of this study is to extend the viable preservation time of hepatic tissues by sub-zero non- freezing (SZNF) storage in a supercooled preservation medium. The central hypothesis of this study relies on two phenomena: 1) that 3-O-methyl-glucose (3OMG) lowers achievable stable SZNF temperature without major toxic side effects, and that 2) rewarming by normothermic perfusion reduces reperfusion damage. Our hypothesis has been formulated based on our preliminary findings establishing 3OMG as a minimally toxic cryoprotectant for hepatocytes, and establishing that normothermic perfusion can significantly reverse the damaging effects of ischemia. The rationale of the study is that if supercooled preservation can be achieved while avoiding antifreeze toxicity, then organ metabolism can be further slowed thereby reducing anoxic/ischemic damage to minimal levels. Establishment of a sub-zero nonfreezing preservation technology will be a welcome innovation to the field. The work described herein will help develop this enabling technology of supercooled storage, and also establish quantitative standards for evaluating the liver and bioartificial organ viability following preservation. While we focus on the liver, we expect that the protocols established here will also serve as the basis for subzero nonfreezing preservation of other tissue engineered products, such as artificial organ substitutes and seeded scaffold constructs.