Ice-free vitrification and nano warming technology for banking of cardiovascular structures.

摘要 这项建议侧重于通过玻璃化冷冻来翻译无冰冷冻保存，采用一种新的方法 在交变电磁场中使用纳米铁纳米武器进行的体积加热。玻璃化， 在玻璃化转变温度以下的零度以下储存在“玻璃态”而不是晶态冻结相中，是 一种避免结冰的冷冻保存方式。通过快速冷却可实现玻璃化。 材料达到低温储存温度，在那里不能形成冰。最后可以保持营养 通过快速将组织重新加热到高于冰点温度的低温方案 可能会发生成核。维持生命力所需的复温率要高得多。 比在第一个地方实现它所需的冷却速度的大小。最常见的 实现所需冷却和复暖率的方法是基于对流的边界增暖 样品的表面暴露在温度控制的环境中，如液浴。 由于传热的基本原理，在表面边界加热的情况下有尺寸限制。 超过这个范围，就不能阻止样品中心的结晶。此外，由于 固体力学的基本原理，也有一个尺寸限制，超过这个限制的热膨胀 标本可能会导致结构损坏和骨折。纳米级加热过程中的体积加热 低温协议的复温阶段可以缓解这些大小限制。玻璃化已经是一种 重要的促进生殖医学的方法，有可能允许储存和运输 用于各种生物医学用途的细胞、组织和器官。不幸的是，实际应用 由于扩散和阶段的原因，玻璃化一直局限于较小的系统，如细胞和薄组织 改变限制血管、较大组织和器官使用的限制。要绕过这个问题 在我们的初步研究中，我们证明了纳米武器有效地使血管复温。我们的 实验证明，这种创新的复温技术使玻璃化的股动脉和颈动脉复温。 动脉的体积从1毫升到50毫升不等，并保留了细胞活力和生理功能。 然而，厚动脉的升温并不是最理想的。我们建议使用大动物血管，模型 使用体外和体内相结合的方法进一步优化和评估纳米武装血管 学习。在第一阶段，我们将针对一个特定的目标，优化厚壁动脉的无冰玻璃化冷冻， 主动脉和肺，目标是达到90%的生存能力，进展到第二阶段。 第二阶段的特定目标，我们建议使用体外和体内相结合的猪血管模型 学习。磁性纳米颗粒将分布在血管的内部空间周围和内部。 较大的管腔空间使其成为优化玻璃化冷冻和冷冻的良好选择 纳米武器。在目标1中，我们将评估实时运输后的冷冻保存的动脉，比较方法 以及验证基于没有组织开裂而最终批准的运输条件。在AIM 2我们将表征保存至少2年的动脉在无冰冷冻保存后的状态。在……里面 此外，在这个目标中，我们将表征无冰的化学和生物材料特性 冷冻保存的血管。将使用冷冻显微镜来检测冰，以评估有效的玻璃化 拉曼光谱分析形成和冷冻保护剂残留物。在目标3中，我们将执行短期 两种猪血管模型(股动脉和肺动脉入颈动脉)的移植研究(28天) 和肺)，以便验证我们的技术，用于未来的IIb阶段SBIR提案 临床相关的临床前非人类灵长类动物模型和人体组织。

ABSTRACT This proposal focuses on translation of ice-free cryopreservation by vitrification employing a novel approach of volumetric heating by nanowarming using Fe nanoparticles in an alternating electromagnetic ?eld. Vitrification, sub-zero storage below the glass transition temperature in a “glassy” rather than a crystalline frozen phase, is a form of cryopreservation that avoids ice formation. Vitri?cation can be achieved by quickly cooling the material to cryogenic storage temperatures, where ice cannot form. Vitri?cation can be maintained at the end of the cryogenic protocol by quickly rewarming the tissue to temperatures above the temperatures where ice nucleation may occur. The magnitude of the rewarming rates necessary to maintain vitri?cation is much higher than the magnitude of the cooling rates that are required to achieve it in the ?rst place. The most common approach to achieve the required cooling and rewarming rates is by convection based boundary warming in which the the specimen's surface is exposed to a temperature controlled environment, such as a fluid bath. Due to the underlying principles of heat transfer, there is a size limit in the case of surface boundary heating beyond which crystallization cannot be prevented at the center of the specimen. Furthermore, due to the underlying principles of solid mechanics, there is also a size limit beyond which thermal expansion in the specimen can lead to structural damage and fractures. Volumetric heating by nanowarming during the rewarming phase of the cryogenic protocol can alleviate these size limitations. Vitrification is already an important enabling approach for reproductive medicine with the potential to permit storage and transport of cells, tissues and organs for a great variety of biomedical uses. Unfortunately, practical application of vitrification has been limited to smaller systems such as cells and thin tissues due to diffusive and phase change limitations that preclude use for blood vessels, larger tissues and organs. To circumvent this problem we demonstrated that nanowarming effectively rewarms blood vessels in our preliminary research. Our experiments demonstrated that this innovative rewarming technique rewarmed vitrified femoral and carotid arteries in volumes ranging from 1 to 50mL with retention of cell viability and physiologic function. However, warming of thick arteries was suboptimal. We propose using large animal blood vessel, models for further optimization and evaluation of nanowarmed vessels using a combination of in vitro and in vivo studies. In Phase 1 in a single specific aim we will optimize ice-free vitrification of thick walled arteries, aorta and pulmonary, with a go/no go objective of achieving > 90% viability for progression to Phase 2. In Phase 2 specific aims, we propose using porcine vascular models in a combination of ex vivo and in vivo studies. The magnetic nanoparticles will be distributed around and within the internal spaces of vessels. The large vessel lumen space makes them a good choice for optimization of vitrification and nanowarming. In Aim 1 we will evaluate cryopreserved arteries after real time shipping, comparing methods and validating the transport conditions that are finally approved based upon absence of tissue cracking. In Aim 2 we will characterize the post-ice-free cryopreservation state of arteries preserved for at least 2 years. In addition, during this aim we will characterize the chemistry and biomaterial properties of ice-free cryopreserved blood vessels. Effective vitrification will be evaluated using cryomacroscopy to detect ice formation and cryoprotectant residuals by Raman spectroscopy. In Aim 3 we will perform short-term transplant studies (28 days) in two porcine vascular models (femoral and pulmonary artery into the carotid and pulmonary, respectively) in order to validate our technology for a future Phase IIb SBIR proposal using clinically relevant preclinical non-human primate models and human tissues.