Isochoric Pressure Based Preservation of Cells, Tissues and Organs

项目摘要/摘要 在移植和更广泛的生物医学界以及政府内部都得到了广泛的认可 资助机构，需要扩大和改善生物材料的保存，以满足广泛的 致力于生物医学、医学研究和药物开发、器官和组织移植、基于细胞的 治疗、生育和再生医学、应急准备和创伤护理。现行的标准是 器官和血管复合移植(VCA)的保存包括几个小时的低温静态保存 在UW溶液中冰上储存。这导致器官/VCA短缺和丢弃率增加，加剧了 供受者配型不佳导致的缺血性损伤和移植排斥反应，并降低生活质量 对移植受者来说。为了应对这些挑战，我们开创了一种新的热力学 基于稳定平衡状态和等容(恒容)的生物保存方法 在零度以下(-5摄氏度至-20摄氏度)的高温条件下，器官将得到有效保存 和VCA，与目前的临床实践相比，储存时间增加了20倍-28倍(低温6-12小时 储存)，同时避免细胞损伤和在深低温下储存带来的其他挑战。 重要的是，我们将在第一阶段成功展示可行性的基础上，以及Sylvatica的结果 和加州大学伯克利分校合作展示了高度零下等容保存：(1) 支持人体细胞在等容条件下存活的保存鸡尾酒的生物相容热力学压力分布 存活率在75%以上的系统，(2)大鼠全心和全皮肤等容保存的初步扩大成功 在前所未有的温度下(-8摄氏度，-10摄氏度)，以及(3)鱼肌肉和整个生物体的等量保存( 研究模型线虫)。因此，这个第二阶段提案的目标是演示，使用动物和 VCA代表性组织和肢体保存的人体模型，延长VCA的冷冻保存时间 储存和恢复后5-7天和7周或更长时间，有良好的功能结果。在五个具体目标上， 我们将首先使用VCA的皮肤和血管模型来进行冷冻鸡尾酒和方案优化，中央 在积极抑制新陈代谢的同时，实现高负温等容保存的目标 压力耐受性。一种基于计算机的恒容保存平台(腔体、压力/温度传感器) 在第一阶段中使用的成功系统将被设计和建造以支持低温冷冻协议验证 临床测量人体皮肤和血管的大小，然后建立大鼠前肢保存模型，并通过 假性移植，然后进行同种异体原位移植，并对上肢进行全面鉴定 肢体功能和行为恢复。动物保存/移植模型的结果将 用于人体手指和手的高负等容保存的概念验证， 进行全面的失血代谢支持质量评估。

PROJECT SUMMARY/ ABSTRACT There is wide recognition within the transplant and broader biomedical communities, as well as government funding agencies, that extended and improved preservation of biological materials is needed for a huge range of endeavors in biomedicine, medical research and drug discovery, organ and tissue transplantation, cell-based therapies, fertility and regenerative medicine, emergency preparedness, and trauma care. The current standard of preservation for organs and vascular composite allografts (VCAs) consists of few hours of hypothermic static storage in UW solution on ice. This contributes to organ/VCA shortage and increased discard rates, exacerbates ischemic injury and graft rejection due to suboptimal donor-recipient matching, and diminishes the quality of life for transplant recipients. To address these challenges, we have pioneered a novel thermodynamic approach to biopreservation, based on a stable equilibrium state and isochoric (constant volume) condition, at high subzero (-5°C to -20 °C) temperatures, that will allow effective preservation of organs and VCAs, with a 20x-28x increase in storage duration over the current clinical practice (<6-12h of hypothermic storage) while avoiding cellular injury and other challenges created by storage at deep cryogenic temperatures. Importantly, we will build on the successful demonstration of feasibility in Phase I and the results that Sylvatica and UC Berkeley have collaboratively demonstrated with regard to high subzero isochoric preservation: (1) biocompatible thermodynamic pressure profiles for preservation cocktails that support human cell survival in isochoric systems with viabilities above 75%, (2) successful initial scale-up of isochoric preservation to whole rat hearts and skin at unprecedented temperatures (-8°C, -10°C), and (3) isochoric preservation of fish muscle and an entire organism (the research model C. elegans). Therefore, the objective of this Phase II proposal is to demonstrate, using animal and human models of VCA representative tissues and limb preservation, prolonged cryopreservation of VCAs for 5-7 days and weeks, or longer, with good functional outcome post storage and recovery. Across five specific aims, we will first employ skin and vascular models of VCAs for cryostasis cocktail and protocol optimization, with the central goal of enabling high subzero isochoric preservation while actively suppressing metabolism and enhancing stress tolerance. An isochoric preservation platform (chamber, pressure/temperature sensors) based on the successful system used in Phase I will be designed and built to support cryostasis protocols validation using clinical size human skin and blood vessels, then a model of rat forelimb preservation and assessment through pseudo transplantation, and then by orthotopic allotransplantation with comprehensive characterization of upper extremity functional and behavioral recovery. The results from the animal preservation/transplantation model will be used for the proof-of-concept for high subzero isochoric preservation of human fingers and hands, with full exsanguinous metabolic support quality assessment.