Extended Storage of Tissues and Organs in Subzero Environments

摘要 美国目前有大约10万名等待器官移植的患者，这个数字 超过了可用器官的供应，而且还在以每年约5%的速度增长。最有希望的 解决方案、生物人工组织和器官构建以及供体器官重建方法，都是 最终受到生物保存技术的限制，就像实验室中制备的任何组织工程产品一样 将必须存储一段时间才能使用。现行的整个器官的黄金标准 保存是指在冰上冷藏长达72小时，在此期间器官不断变质。一个 超越目前限制的延长组织保存时间的更好的生物保存方法尚未成为 发展起来的。这种方法将为组织和器官保存提供关键的使能技术， 组织和器官运输，以及组织和器官移植。 本研究的目的是为了延长肝组织的存活保存时间，通过在零度以下的非 冷冻(SZNF)在过冷保存介质中的存储。这项研究的中心假设依赖于 两个现象：1)3-O-甲基葡萄糖(3OMG)降低了可实现的稳定的SZNF温度 主要毒副作用；2)常温灌流复温可减少再灌流损伤。我们的 根据我们的初步发现，提出了一种假设，将3OMG确定为毒性最低的 肝细胞的冷冻保护剂，并建立常温灌流可以显著逆转 缺血的破坏性影响。这项研究的基本原理是，如果能够实现过冷保存 在避免防冻剂毒性的同时，可以进一步减缓器官新陈代谢，从而减少 缺氧/缺血损伤降至最低水平。 建立零度以下的不冻保存技术将是该领域的一项受欢迎的创新。 这里描述的工作将有助于开发这种过冷存储的使能技术，并且还 建立评价保存后肝脏和生物人工器官活性的量化标准。 虽然我们将重点放在肝脏上，但我们预计这里建立的方案也将作为 其他组织工程产品的零度非冷冻保存，如人造器官替代品和 种子脚手架构造。

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.