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

摘要 该提案的重点是采用玻璃化的无冰冷冻保存的翻译， 体积加热，通过使用铁纳米粒子在交变电磁？eld.玻璃化， 在低于玻璃化转变温度的温度下，以“玻璃态”而不是结晶冷冻相进行零度以下储存， 一种避免结冰的低温保存方法。维特里？阳离子可以通过快速冷却 材料的低温储存温度，在那里冰不能形成。维特里？阳离子可以保持在末端 通过快速将组织重新加热到高于冰的温度， 可能发生成核。维持玻璃体所需的复温率的大小？阳离子要高得多 而不是冷却速率的大小，这是实现它所需要的？第一个地方。最常见的 实现所需冷却和复温速率的方法是通过基于对流的边界升温， 其中试样的表面暴露于温度受控的环境，例如流体浴。 由于传热的基本原理，在表面边界加热的情况下存在尺寸限制 超过该值就不能防止试样中心处的结晶。而且因为 根据固体力学的基本原理，也存在尺寸限制，超过该尺寸限制， 可能导致结构损伤和断裂。在加热过程中， 低温方案的复温阶段可以减轻这些尺寸限制。玻璃化已经是一种 生殖医学的重要有利方法，有可能允许储存和运输 细胞、组织和器官用于各种生物医学用途。不幸的是， 玻璃化由于扩散性和阶段性而局限于较小的系统，例如细胞和薄组织。 变更限制，排除用于血管、较大组织和器官。为了避免这个问题 在我们的初步研究中，我们证明了复温能有效地使血管复温。我们 实验表明，这种创新的复温技术复温了玻璃化的股骨和颈动脉， 体积范围为1至50 mL的动脉，保留细胞活力和生理功能。 然而，厚动脉的加温是次优的。我们建议使用大型动物血管模型， 为了使用体外和体内的组合进一步优化和评估带臂血管， 问题研究在第一阶段，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， 主动脉和肺动脉，进行/不进行的目标是实现> 90%的进展到2期的生存力。在 第2阶段的具体目标，我们建议使用猪血管模型在体外和体内相结合 问题研究磁性纳米颗粒将分布在血管的内部空间周围和内部空间内。 大的血管腔空间使它们成为优化玻璃化和冷冻的良好选择。 令人惊讶。在目标1中，我们将在真实的运输后评估冷冻保存的动脉，比较方法 以及验证基于没有组织破裂而最终批准的运输条件。在Aim中 2我们将描述保存至少2年的动脉的无冰冷冻保存后的状态。在 此外，在此期间，我们将表征无冰的化学和生物材料特性， 冷冻保存的血管有效的玻璃化将评估使用低温显微镜检测冰 通过拉曼光谱法测定形成和冷冻保护剂残留。在目标3中，我们将执行短期 在两个猪血管模型（股动脉和肺动脉进入颈动脉）中的移植研究（28天 和肺），以验证我们的技术用于未来的IIb期SBIR提案， 临床相关的临床前非人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.