Subzero preservation of vascular composite allografts

摘要。20年前证实了VCA移植的技术可行性， 结果令人鼓舞。然而，终身免疫抑制的伦理计算通常排除VCA 移植为了避免长期免疫抑制的副作用，最有希望的方法是诱导 通过骨髓共移植的混合嵌合体耐受。这是唯一一种在以下方面显示出有效性的策略： 实体器官移植的大动物和临床研究。然而，目前临床试验中的耐受性方案要求 从最初的供体移植物恢复到受体，大约需要3到7天的时间来预处理受体 移植回避预处理诱导混合嵌合体的需要似乎不容易实现， 在临床VCA移植病例中，一个团队尝试失败。与肾脏相反，在VCA中， 活体捐赠，使预先接受者条件治疗在临床上不现实。因此，一种技术， 保存以连接受体条件化的时间仍然是实现耐受诱导的关键必要条件 在VCA移植中。 我们的总体目标是开发一种高低温保存协议，将延长血管组织的活力 在移植前的保存期间，从而创建实施耐受诱导方案的时间窗。 这项研究的首要假设是，在低温保护剂（CPA）加载期间减轻渗透冲击， 减少加载和卸载CPA期间对内皮细胞的剪切损伤和毒性，以及不受控制的旁路 在通过冷冻从零度以下温度复温期间的缺血，将使得能够上级和延长保存 同种异体肢体移植这一假设是根据我们先前的数据制定的，这些数据表明， 人类肝脏显示出可扩展性的证据;然而，最初尝试直接采用大鼠和猪肢体的相同方案 导致过度水肿，并且与对照组相比， 肝脏为这项工作建立的多机构伙伴关系汇集了两个机构的独特专长： 1)MGH医学与外科工程中心的零度以下器官储存，以及2）明尼苏达大学 医学工程研究所的独特技术。 我们的预期贡献是在这里的发展，一个新的保存协议，大鼠血管复合组织 这将总储存时间从数小时延长到数天，并提供时间使受体准备诱导耐受性。这 这一贡献是重要的，因为这里开发的方法可能是临床协议的概念验证。 在VCA移植中的耐受性诱导是实用的，因此可以使其广泛使用，这是不可能的。 目前。

ABSTRACT. The technical feasibility of VCA transplantation was demonstrated 20 years ago and the initial clinical outcomes have been encouraging. However, the ethical calculus of lifelong immunosuppression does often preclude VCA transplantation. To avoid the side effects of long-term immunosuppression, the most promising approach is the induction of tolerance by mixed chimerism via bone-marrow co-transplantation. This is the only strategy that has shown efficacy in large animal and clinical studies of solid organ transplantation. However, current tolerance protocols in clinical trials require somewhere between 3 to 7 days to pre-condition the recipient, measured from initial donor graft recovery to recipient transplantation. Sidestepping the need for preconditioning to induce mixed chimerism does not appear easy to achieve and failed in a clinical VCA transplant case when attempted by one team. Contrary to kidneys, in VCA there is no possibility of living donation, making advance recipient conditioning clinically unrealistic. Therefore, a technology to extend graft preservation to bridge the time for recipient conditioning remains a critical necessity that would enable tolerance induction in VCA transplants. Our overall goal is to develop a high subzero preservation protocol that will prolong the viability of vascular tissues during preservation prior to transplantation, thereby creating a time window to implement tolerance induction protocols. The overarching hypothesis of this study is that alleviating osmotic shocks during cryoprotective agent (CPA) loading, reducing shear injury and toxicity to endothelial cells during loading and unloading CPAs, and bypassing uncontrolled ischemia during rewarming from subzero temperatures by nanowarming, will enable superior and extended preservation of limb allografts. This hypothesis has been formulated based on our prior data demonstrating success in supercooling of human livers showing proof of scalability; however initial attempts to directly adopt the same protocol for rat and pig limbs led to excessive edema and identification of extreme sensitivity to ischemic injury and vascular resistance compared to livers. The multi-institutional partnership assembled for this work brings together the unique expertise of two institutions: 1) subzero organ storage at the MGH Center for Engineering in Medicine & Surgery, and 2) the University of Minnesota Institute for Engineering in Medicine’s unique technology of nanowarming. Our expected contribution here is the development of a new preservation protocol for rat vascularized composite tissues that extends total storage duration from hours to days, and provides time to prepare the recipient to induce tolerance. This contribution is significant because the methods developed here could be the proof-of-concept for a protocol that is clinically practical for tolerance induction in VCA transplants, and could therefore enable their wide-spread use which is not possible currently.