Organ banking for transplant--kidney cryopreservation by vitrification and novel nanowarming technology

摘要 慢性肾脏疾病是一个重大的医疗保健问题，影响着15%的美国人口，耗资数十亿美元 以每年医疗保健美元计。移植是大多数进展性疾病患者的最佳选择， 因此，与透析相比，预期寿命显著延长，生活质量得到改善。这个 据估计，美国潜在的已故捐赠者器官供应量将超过目前移植的器官数量 增加4到5倍，可接受移植的器官数量的主要限制是 从康复到植入期间所遭受的缺血性损伤。一种预先冷冻保存或“储存”肾脏的方法 移植将有效地消除时间对器官分配供应链的影响。这 将允许一种新的移植范例，这将改善供者/受者匹配，允许更好的 患者准备，促进耐受诱导方案，提高器官利用率，同时改善移植物 和病人的生存。克服传统策略局限性的一种有希望的方法是 玻璃化--即冷却器官的速度太快，以至于它们无法经历从液体到固体的相变 冰。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 从理论上讲，存储是无限期的。然而，关键的挑战是在不结冰或不破裂的情况下重新升温： 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。在.期间 在我们最初的R01资助下，我们开发了一种名为“纳米武器”的新方法，实现了这两个目标。 将氧化铁纳米颗粒与CPA溶液一起灌流到器官的整个血管系统。这个 然后通过冷却将器官玻璃化，并按需将其放入射频线圈中复温，以诱导 在纳米颗粒中加热，因此，从器官内部加热。我们发现纳米武器可以复温 动物模型中的玻璃化器官，包括肾脏。我们最近首次证明，纳米武器 器官移植后，在体外和体内都能发挥作用。此外，我们还展示了成功的玻璃化冷冻和 人类(猪)大小的肾脏的纳米温控。这些新的数据支持我们的方法的可行性 冷冻保存和纳米机械臂整个人体器官用于移植。尽管如此，许多问题 仍然存在，包括纳米武器肾脏与对照器官相比功能如何，如果有的话，发生了什么损伤 以及如何扩大到人体大小的器官。在本次续订R01中，我们建议：(1) 定量评估冷冻保存和纳米武器肾移植大鼠模型的功能，包括长期肾移植 长期保存、长期功能、损伤模式和宿主免疫反应的改变，(2)工程师 并优化放大纳米武器玻璃化人体大小的器官，以及(3)玻璃化和纳米手臂人体大小 同时测量肾脏的活力、结构完整性和器官功能。

ABSTRACT Chronic kidney disease is a significant healthcare issue affecting >15% of the U.S. population and costing billions in healthcare dollars annually. Transplantation is the best option for most patients with progressive disease, resulting in a significant increase in life expectancy and improved quality of life compared to dialysis. The potential U.S. deceased donor organ supply is estimated to exceed the current number of organs transplanted by a factor of 4- to 5-fold, with a major limitation to the number of acceptable organs for transplant being the ischemic injury sustained between recovery and implantation. A method to cryopreserve or “bank” kidneys prior to transplant would effectively remove the influence of time from the supply chain of organ distribution. This would allow a new paradigm for transplantation that would improve donor/recipient matching, allow for better patient preparation, facilitate tolerance induction protocols, and increase organ utilization while improving graft and patient survival. One promising approach that overcomes the limitations of conventional strategies is vitrification—that is, cooling organs so quickly that they 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 viable storage is theoretically indefinite. The critical challenge, however, is rewarming without ice formation or cracking: if rewarming is too slow, ice crystals form, and if rewarming is not uniform, thermal stress causes cracking. During our initial R01 funding, we developed a novel approach termed “nanowarming” that achieved both objectives. Iron oxide nanoparticles were perfused throughout the vasculature of the organ along with CPA solutions. The organ was then vitrified by cooling and rewarmed on-demand by placing it in a radiofrequency coil that induces heating in the nanoparticles and, therefore, from within the organ. We found that nanowarming could rewarm vitrified organs, including kidneys, in animal models. We have recently shown, for the first time, that nanowarmed organs function in vitro and in vivo following transplantation. Further, we showed successful vitrification and nanowarming of human-sized (porcine) kidneys. These new data support the feasibility of our approach to cryopreserve and nanowarm whole human organs for transplantation. Nevertheless, many questions remain, including how nanowarmed kidneys function compared to control organs, what, if any, injury occurs during nanowarming, and how to scale up to human-sized organs. In this renewal R01, we propose to: (1) Quantitatively assess cryopreserved and nanowarmed kidney transplant function in a rat model, including long- term preservation, long-term function, modes of injury, and alterations of the host immune response, (2) Engineer and optimize scale-up for nanowarming vitrified human-sized organs, and (3) Vitrify and nanowarm human-sized kidneys while measuring viability, structural integrity, and organ function.