High subzero preservation of liver for transplantation

摘要 由于等待名单上的患者数量是美国接受捐赠器官患者数量的五倍多， 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制在几个小时内。需要更长的保存时间 启用全球配对计划，消除不必要的器官浪费，并降低与计划外相关的成本 手术，并使基于混合嵌合体的耐受诱导方案成为可能，该方案要求供体器官 保持存活数天，以便可以安全地进行接受者的预适应。因此，此应用程序的重点是 解决移植中一个主要的未得到满足的需求--延长器官可保持“暂停”状态的时间 动画《活体外》。我们的方法的灵感来自于自然界中冬眠和耐寒的动物。我们的目标是拥抱 并控制结冰以在胞外冰存在的情况下实现较高的零下储存温度，并储存 持续时间从几周到最终几个月。我们的假设是，冰在非破坏性冰冻状态下的存在 能够在较低温度(低至-30°C)下存储，从而使代谢停滞更深达数周，同时还 使之能够扩大到人体器官。在之前的资助期间，我们开发了一种新的方法来使用冰核 细菌严密控制结冰温度，并开发了一种低至-15摄氏度的部分冷冻方案，可以 具有体外功能的大鼠肝脏保存长达5天，并在人类肝脏中建立了概念验证。在这 为了竞争续订申请，我们建议在我们成功的基础上再接再厉，将保存温度降至-30°C。 从而实现了长达数周的暂停生命。我们建议(I)控制肝脏中冰的形成，这是我们先前的研究 已被证明是一个关键障碍；(2)制定有针对性的方法，以改善内皮细胞的保存，这些方法在 组织和冰的界面，是损伤的第一个点，以及(Iii)制定一个精心设计的代谢方案，以 最大限度地回收ATP，提高移植移植物的存活率。

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.