New approaches to kidney banking through nature-inspired high sub-zero preservation strategies

摘要 肾移植等待名单占美国器官移植等待名单的83%。然而，每5名患者死亡 或者病得太重而不能移植，3个肾脏被丢弃。器官保存质量与移植物寿命有关 肾移植10年后存活率只有50%。将保存时间延长至一周或更长时间将使 全国范围内(理论上是全球的)供受者配型，允许许多高质量的肾脏今天没有被移植 (例如，扩展标准供体肾脏)提供给最需要它们的患者。肾脏保存时间 7-9天可转化为临床免疫耐受诱导，避免终生 从活体供体移植到已故器官捐赠的免疫抑制要求 (占所有移植的80%)，改善移植受者的生活，并为医疗保健系统节省约1亿美元 每年1美元。改进和扩展的保存技术，以及现成的可用，可以提高 筛查传染病/恶性肿瘤，评估器官质量，移植的药物/基因疗法 功能增强，并支持异种移植研究。此外，保存下来的肾脏的3D切片 移植无与伦比，在药物安全性/有效性测试中具有广泛的适用性，研究课题种类繁多。 在项目第一阶段可行性成功的基础上，展示了史无前例的(兔)肾 保存长达14天，我们在这里扩大这项新技术，以开发(I)受自然启发和 (Ii)启用机器灌流的零度以下溶液，可显著延长人和 猪(异种)肾。使用超稳定平衡应用高零下温度(-12至-20°C) 方法与增强应激耐力和抑制代谢策略相结合。已优化 机器灌注方案，精切肾切片的3D组合高通量筛查， 采用先进的微米CT成像、全人类肾脏和猪存活自体移植模型。 基于第一阶段的重大成就和我们团队的器官保存经验，我们开发了 增强人体肾脏平衡无冰高低温银行二期技术与策略 VIA：1)支持预适应方案的多温度多步机灌流平台，用于临床 鳞片肾脏有效和均匀的渗透，在所有肾脏隔室水平，与(I)生物相容的冷冻 根据毒性中和和相互稀释原则配制的鸡尾酒，以及(Ii)有效 用于增强再灌注的复苏/修复方案；2)验证(168-216小时)7-9天的保存 完全无冰，(Iii)储存肾脏的跨国运输，以及体外肾脏的临床实践 灌流；3)长期存活的猪模型原位肾移植，有无“任何地方” 和4)保存7-9天的人体肾脏的随机生理体外评估试验。 第二阶段为集中保存和管理卓越中心的后勤模式奠定了基础 评估，极大地改善了临床结果和患者在全国和世界各地的就医机会。

ABSTRACT The kidney transplant waitlist comprises 83% of the U.S. organ transplant waitlist. Yet, for every 5 patients who die or become too sick for transplantation, 3 kidneys are discarded. Organ preservation quality is linked to graft lifespan with kidneys having only 50% graft survival after 10 years. Extending preservation to a week, or longer, would enable nationwide (theoretically global) donor-recipient matching, allowing many good quality kidneys not transplanted today (e.g., extended criteria donor kidneys) to be offered to the patients who most need them. Kidney preservation times of 7-9 days would enable translation of clinical immune tolerance induction, and avoidance of life-long immunosuppression requirements, from contexts of living donor transplantation to deceased organ donation (80% of all transplants), improving the lives of transplant recipients, and saving the healthcare system ~ $100 million dollars each year. Improved and extended preservation technology, with off-the-shelf availability, can enhance screening for transmissible diseases/malignancies, allow assessment of organ quality, drug/gene therapies for graft function augmenting, and support xeno-transplantation research. Moreover, 3D slices from preserved kidneys unmatched for transplant, have wide applicability in drug safety/efficacy testing, and a large variety of research topics. Building on the project's Phase I feasibility success that demonstrated unprecedented (rabbit) kidney preservation for up to 14 days, we here scale-up this new technology to develop a (i) nature-inspired and (ii) machine perfusion enabled, subzero solution to dramatically extend preservation times of human and porcine (xeno) kidneys. High subzero temperatures (-12 to -20°C) applied using an ultra-stable equilibrium approach are combined with stress tolerance enhancement and metabolic suppression strategies. Optimized machine perfusion protocols, 3D combinatorial high throughput screening with precision cut kidney slices, advanced µCT imaging, whole human kidney, and porcine survival auto transplantation models are employed. Based upon the significant Phase I achievements and our team's organ preservation experience, we develop technologies and strategies in Phase II for enhanced human kidney equilibrium ice-free high subzero banking via: 1) multi-temperature multi-step machine perfusion platform supportive of pre-conditioning protocols, for clinical scale kidney effective and uniform permeation, at all renal compartmental levels, with (i) biocompatible cryostasis cocktails formulated on the basis of toxicity neutralization and mutual dilution principles, and (ii) effective revival/reconditioning protocols for enhanced reperfusion; 2) validation of (168-216h) 7-9 days preservation in complete absence of ice with (iii) cross country shipping of stored kidneys, and clinical practices of ex vivo renal perfusion; 3) porcine model kidney orthotopic transplantation with long-term survival, with and without “anywhere in the world” shipping, and 4) randomized physiologic ex vivo assessment trial of 7-9 days stored human kidneys. This Phase II sets the stage for logistical model of centralized centers of excellence for preservation and assessment, enabling vastly improved clinical outcomes and patients' access across the country, and world.