High subzero preservation of liver for transplantation

摘要 在等待名单上的病人数量是美国接受捐赠器官的病人数量的五倍多， 移植领域正面临严重的供体短缺危机。尽管进行了数十年的研究，但一个主要的关键瓶颈是 目前整个器官的保存时间限于几个小时的储存。需要更长的保存时间 实现全球匹配计划，消除不必要的器官浪费，并降低与计划外移植相关的成本。 手术，并使基于混合嵌合体的耐受性诱导方案成为可能，该方案要求供体器官 保持存活数天，以便接受者可以安全地进行预处理。因此，本申请的重点是 通过延长器官可以保持在“悬浮”状态的时间长度， 动画”离体。我们的方法受到自然界中冬眠和耐冻动物的启发。我们的目标是拥抱 并控制冰的形成，以在存在细胞外冰的情况下实现高的零下储存温度， 持续数周至最终数月。我们的假设是，在无害的冰冻状态下， 能够在较低的温度（低至-30 ° C）下储存，因此可以在数周内保持更深的代谢停滞，同时还 使其能够扩大到人体器官。在上一个资助期，我们开发了一种新的方法， 细菌来严格控制冰的形成温度，并开发了一种低至-15 ° C的部分冷冻方案， 将大鼠肝脏储存长达5天，具有离体功能，并且还在人类肝脏中建立了概念验证。在这 竞争更新应用，我们建议在我们成功的基础上，将保存温度降至-30 ° C， 因此可以假死数周。我们建议（i）控制肝脏中的冰形成，这是我们先前的研究 被证明是一个关键的障碍;（ii）开发有针对性的方法，以改善内皮细胞的保护，这是在 组织和冰的界面，是第一个受伤点，以及（iii）制定一个精心设计的代谢方案， 最大化ATP回收并提高移植物的存活率。

ABSTRACT With more than five times the number of patients on the wait list than will receive a donor organ in the USA, the field of transplantation is facing a serious donor shortage crisis. Despite decades of research, a major critical bottleneck is the current preservation times for whole organs are limited to few hours of storage. Longer preservation times are required to enable global matching programs, eliminate unnecessary waste of organs, and reduce costs associated with unplanned surgeries, and are enabling for mixed-chimerism based tolerance induction protocols which require the donor organ to be kept viable for days so that recipient preconditioning can be done safely. Consequently, the focus of this application is to address a major unmet need in transplantation by prolonging the length of time organs can be kept in “suspended animation” ex vivo. Our approach is inspired by the hibernating and freeze-tolerant animals in nature. We aim to embrace and control the ice formation to achieve high subzero storage temperatures in the presence of extracellular ice, and storage durations of weeks to ultimately months. It is our hypothesis that the presence of ice in a non-injurious frozen state enables storage at lower temperatures (down to -30°C) and consequently a deeper metabolic stasis for weeks, while also enabling scale-up to human organs. In the previous funding period, we developed a new method to use ice nucleating bacteria to tightly control ice formation temperature and developed a partial freezing protocol down to -15°C that can store rat livers up to 5 days with ex vivo function, and also established a proof-of-concept in human livers. In this competing renewal application, we propose to build on our success and reach preservation temperatures down to -30°C, thus achieving suspended animation for weeks. We propose (i) to control ice formation in liver, which our prior studies demonstrated to be a key barrier; (ii) to develop targeted approaches to improve preservation of endothelium, which are at the interface of tissue and ice and are the first point of injury, and (iii) to develop a choreographed metabolic protocol to maximize ATP recovery and improve viability of the grafts for transplant.