Cryopreservation and nanowarming enables whole liver banking for transplantation, cell therapy and biomedical research

摘要 近三分之一的已故供体肝脏未用于移植或其他目的。这些器官中的许多 如果能延长保存时间，将对治疗和研究应用具有重要价值。 超低温(&lt；-140°C)的低温保存可以实现器官的无限期保存。之前的尝试 由于冰晶形成造成细胞和结构的破坏，器官冷冻保存失败。一 克服传统方法局限性的有希望的方法是玻璃化--即冷却 器官的速度如此之快，以至于器官内的水不能经历从液体到固体冰的相变。 在冷冻保护剂(CPA)的帮助下，器官进入稳定的玻璃状状态，在这种状态下，活的 理论上，储存时间是无限的。然而，关键的挑战是在不结冰的情况下重新变暖或 破解。如果复温太慢，就会形成冰晶；如果复温不均匀，热应力就会导致破裂。 因此，升温的速度和一致性是至关重要的。我们已经开发出一种新的复温方法 被称为“纳米武器”，实现了这两个目标。氧化铁纳米颗粒被灌流到整个 器官的血管构筑和CPA溶液。然后，器官可以通过冷却(现有的 技术)，并根据需要通过将其放置在射频线圈中进行复温，该线圈在 纳米颗粒，因此，从器官内部。在初步研究中，我们第一次表明 我们可以玻璃化和纳米化人类大小的(如猪)和大鼠的肝脏，从而避免结冰或破裂 并保持生存能力和器官水平的功能。在这一身体上的成功之后，我们在这里提出第一个 评估这些纳米武装器官的移植和生物活性/功能成功的研究。我们的 中心假设是，通过玻璃化和纳米武器进行冷冻保存将使功能长期 用于移植、治疗和生物医学研究的完整人类肝库。虽然我们的长期 我们的目标是开发一种用于移植的人体肝脏冷冻保存方法，我们这个项目的目标是改进 全肝保存技术：1)提高保存大鼠肝脏的体内外功能 在常温灌流和移植模型中，2)确定细胞应激、损伤和 肝脏冷冻保存造成的死亡和减轻损伤的策略，以及3)提供进一步的调查 大动物(猪)和人类肝脏的冷冻保存和复温。如果成功，这种方法可能会 彻底改变这些宝贵资源的分配和利用方式，为患者和社会造福。

ABSTRACT Nearly one-third of deceased donor livers are unused for transplant or other purposes. Many of these organs would be valuable for therapeutic and research applications if preservation times could be extended. Cryopreservation at ultralow temperatures (< -140°C) can enable indefinite organ storage. Previous attempts at organ cryopreservation have failed due to cellular and structural disruption caused by ice crystal formation. One promising approach that overcomes the limitations of conventional strategies is vitrification – that is, cooling organs so quickly that the water within the organ cannot undergo the phase transition from liquid to solid ice. With the help of cryoprotective agents (CPAs), the organ enters a stable glass-like state wherein the viable storage time is theoretically unlimited. The critical challenge, however, is rewarming without ice formation or cracking. If rewarming is too slow, ice crystals form; if rewarming is not uniform, thermal stress causes cracking. Hence, speed and uniformity of warming are essential. We have developed a novel rewarming approach termed “nanowarming” that achieves both objectives. Iron oxide nanoparticles are perfused throughout the vasculature of the organ along with CPA solutions. The organ can then be vitrified by cooling (an existing technology) and rewarmed as needed by placing it in a radiofrequency coil that induces heating in the nanoparticles and, therefore, from within the organ. In preliminary studies, for the first time we have shown that we can vitrify and nanowarm human sized (i.e. porcine) and rat livers, thereby avoiding ice formation or cracking and preserving viability and organ-level function. Following on this physical success, we propose here the first study to assess both transplantation and biological viability/function success of these nanowarmed organs. Our central hypothesis is that cryopreservation by vitrification and nanowarming will enable functional long-term whole human liver banking for transplant, therapeutic and biomedical research purposes. While our long-term goal is to develop a method for cryopreserving human livers for transplant, our goals for this project are to refine whole-liver preservation technology to 1) improve in vitro and in vivo functionality of preserved rat livers during normothermic perfusion and in transplant models, 2) determine the mechanisms of cellular stress, injury, and death resulting from liver cryopreservation and strategies for injury mitigation, and 3) provide further investigation of large animal (porcine) and human liver cryopreservation and rewarming. If successful, this approach could revolutionize how these precious resources are allocated and utilized for patient and societal benefit.