Extended preservation of human livers: a nature inspired, high subzero controlled, limited freezing approach

摘要 在等待接受肝脏移植的患者中，五分之一的患者没有及时得到新的肝脏，而更多的患者从未被列入名单，但有可能 从肝脏置换中获益。而“搭桥移植”技术(心脏辅助装置和透析) 已经改变了心力衰竭和肾衰竭的前景，但对肝脏患者没有这样的治疗方法 失败了。当前肝脏共享限制(约500英里)是基于有限的保存期(6-10小时)。 通过将保存时间延长到几天，我们可以在全国范围内(理论上是全球范围内)实现捐赠者-接受者 匹配，允许许多今天未移植的肝脏(例如，扩展标准供体肝脏的子集， 已被证明提供了大量的生存福利)，以提供给最需要它们的患者。 移植外科医生对这一可能性表示兴奋，并认为这一成就 会对肝脏的等待死亡率产生深远的影响。除了其他好处外，这还可以增加贪污 延长寿命并减少免疫排斥反应，消除对昂贵的飞机和直升机运输的需要，转变 肝移植的实践从急诊手术转变为灵活安排的手术，并缓解了 在肝移植方面存在严重的差异。它还将允许免疫耐受诱导方案，现在已经 在活体捐赠方面取得成功，以适应已故捐赠者的情况。为了满足这些需求，我们 将开发一个全面的人体肝脏银行系统，供移植使用5-7年 延长目标为10天或更长时间的天数。我们创建了一种综合的双管齐下的方法来 为肝脏保存的关键阶段开发新的瘀血鸡尾酒：预适应和保护 (储存前)、保存(储存期间)和复苏-复苏-修复(储存后)。同时，我们 正在开发先进的灌流、储存和器官评估设备，以便在移植决策之前进行评估。我们的 Approach采用了最好的耐寒和冬眠物种的策略，用最近的 科学和生物工程的进步。重要的是，我们并不寻求解决古典音乐的所有问题 超低温保存，但却是第一个在高零下发展热力学平衡保存的人 (HSZ)温度(-10°C至-30°C)，结合程序性代谢抑制和增强的应激 宽容。这种方法的可行性已经得到证明；我们已经将老鼠的肝脏储存了5天 在HSZ温度下，并成功地在-15℃保存了第一个完整的人类肝叶。 计划中，第1阶段的目标将是进一步完善我们的协议并验证我们最初前所未有的存储 大鼠肝脏保存5-7天后使用综合保存机保存 血流灌注与功能评估。我们还将扩展到整个人类肝叶，以及随后的肝细胞 与世隔绝。第二阶段将专注于优化影响大鼠和人类肝脏保存结果的关键参数。 我们的目标是通过(I)对冰生长的最佳调节，(Ii)自然解毒/恢复活力，实现6-10天以上的储存 灌流阶段和(Iii)使用生物热调制器原型装置的热力学精确控制。

ABSTRACT 1 in 5 liver waitlist patients do not receive a new liver in time, while many more patients are never listed but could benefit from liver replacement. While “bridge-to-transplant” technologies (ventricular assist devices and dialysis) have transformed the outlook for heart failure and kidney failure, no such treatment exists for patients with liver failure. Current liver sharing limits (approx. 500 miles) are based on limited preservation durations (6-10 hours). By extending preservation to just a few days we can enable nationwide (theoretically global) donor-recipient matching, allowing many livers that go untransplanted today (e.g. subsets of extended criteria donor livers that have been shown to offer substantial survival benefits) to be offered to the patients who most need them. Transplant surgeons have expressed excitement about this possibility, and it is thought that this achievement would have a profound impact on liver waitlist mortality. Among other benefits, this can also increase graft lifespan and reduce immunological rejection, eliminate the need for costly jet and helicopter transport, transform the practice of liver transplantation from emergency surgery to a flexibly scheduled procedure, and mitigate the severe disparities in liver transplantation. It will also allow immune tolerance induction protocols that have now achieved success in living donation to be adapted to the deceased donor context. To meet these needs, we will develop a comprehensive system for banking human livers for transplantation for periods of 5-7 days with a stretch goal of 10+ days or longer. We have created an integrated two-pronged approach to develop new stasis cocktails optimized for the critical phases of liver preservation: preconditioning and protection (prior to storage), preservation (during storage) and revival- resuscitation-repair (after storage). In parallel, we are creating devices for advanced perfusion, storage, and organ assessment before transplant decision. Our approach adapts the best strategies of freeze-tolerant and hibernating species, augmenting them with recent scientific and bioengineering advances. Importantly, we do not seek to solve all the problems of classical cryopreservation, but rather to be the first to develop preservation in thermodynamic equilibrium at high subzero (HSZ) temperatures (-10°C to -30°C), combined with programmed metabolic depression and enhanced stress tolerance. Feasibility of this approach has already been demonstrated; we have banked rat livers for 5 days at HSZ temperatures, and successfully preserved the first whole human liver lobe at -15°C. For this fast-track proposal, the Phase 1 goal will be to further refine our protocols and validate our initial unprecedented storage duration by banking rat livers for 5-7 days and using comprehensive post-preservation normothermic machine perfusion with functional assessment. We will also scale to whole human liver lobes, with subsequent hepatocyte isolation. Phase 2 will focus on optimizing key parameters that affect preservation outcomes in rat and human livers. Our goal is 6-10+ days of storage via (i) optimal modulation of ice growth, (ii) nature-inspired de-toxify/reenergize perfusion phase and (iii) precise control of thermodynamics using a prototype Bio-Thermodulator device.