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

用于心血管结构银行的无冰玻璃化和纳米加温技术。

基本信息

批准号：
10026454
负责人：
Kelvin G.M. Brockbank
金额：
$ 83.78万
依托单位：
TISSUE TESTING TECHNOLOGIES, LLC
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-11-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10026454
关键词：
Achievement Animal Model Animals Aorta Area Arteries Bathing Biocompatible Materials Biological Biological Assay Biomechanics Blood Preservation Blood Vessels Blood Volume Cardiovascular system Carotid Arteries Cations Cell Survival Cells Chemistry Clinical Controlled Environment Convection Coronary Artery Bypass Cryopreservation Cryopreserved Tissue Crystallization Detection Diagnostic Dialysis procedure Diffuse Disaccharides Dry Ice Electromagnetics Endothelium Engineering Evaluation Excision Exposure to Extracellular Matrix Family suidae Formulation Fracture Freezing Fresh Tissue Future Generations Glass Heating Histopathology Human Hyperplasia Ice In Vitro Inflammation Laser Scanning Microscopy Lead Liquid substance Logistics Lung Magnetic Resonance Imaging Magnetic nanoparticles Market Research Measurement Mechanics Medial Medicine Metals Methods Microscopic Modeling Nitrogen North America Organ Outcome Patients Peripheral Permeability Phase Physiological Property Protocols documentation Pulmonary artery structure Raman Spectrum Analysis Regenerative Medicine Reproductive Medicine Research Residual state Rewarming Sample Size Sampling Scanning Electron Microscopy Shipping Small Business Innovation Research Grant Solid Specimen Structure Surface System Techniques Technology Temperature Testing Thick Thinness Time Tissue Engineering Tissue Preservation Tissue Viability Tissues Transition Temperature Translations Transplantation Vascular Graft Work base blood vessel transplantation cellular engineering clinical application clinically relevant cryogenics crystallinity cytotoxicity experience experimental study femoral artery human tissue in vivo innovation method development microwave electromagnetic radiation nano nanoparticle nanowarming nonhuman primate novel novel strategies packaging material phase change physical state practical application pre-clinical preclinical evaluation preservation pressure prevent radio frequency resazurin response second harmonic technology development transplant model trauma care vapor

项目摘要

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.

抽象的该提案的重点是采用一种新颖的方法通过玻璃化转化无冰冷冻保存在交变电磁场中使用铁纳米粒子进行纳米加热体积加热，在“玻璃态”而不是结晶冷冻相中低于玻璃化转变温度的零下存储是一种避免结冰的冷冻保存形式。材料达到无法形成冰的低温储存温度。通过快速将组织重新加热到高于冰的温度的低温协议可能会发生成核。维持玻璃化所需的复温速率要高得多。首先，比实现它所需的冷却速率的大小要大。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样品的表面暴露在温度受控的环境中，例如液浴。由于传热的基本原理，表面边界加热存在尺寸限制超过这个值，就无法阻止样品中心的结晶。固体力学的基本原理，也存在尺寸限制，超过该尺寸限制，样品在纳米加热过程中可能会导致结构损坏和断裂。低温方案的复温阶段可以缓解这些尺寸限制。生殖医学的重要支持方法，有可能允许储存和运输不幸的是，细胞、组织和器官具有多种生物医学用途。由于扩散和相的原因，玻璃化仅限于较小的系统，例如细胞和薄组织改变无法用于血管、较大组织和器官的限制，以避免这个问题。我们在初步研究中证明，纳米变暖可以有效地使血管重新变暖。实验表明，这种创新的复温技术可以复温玻璃化的股骨和颈动脉体积从 1 到 50mL 的动脉，保留细胞活力和生理功能。然而，粗动脉的变暖并不理想，我们建议使用大型动物血管模型。结合体外和体内进一步优化和评估纳米加热血管在第一阶段的单一特定目标中，我们将优化厚壁动脉的无冰玻璃化，主动脉和肺动脉，进行/不进行的目标是达到 > 90% 的生存率，进入第 2 阶段。第二阶段的具体目标，我们建议结合离体和体内使用猪血管模型研究中，磁性纳米粒子将分布在血管内部空间周围和内部。较大的容器内腔空间使其成为优化玻璃化和在目标 1 中，我们将评估实时运输后的冷冻动脉，比较方法。并验证基于不存在组织裂纹而最终批准的运输条件。 2 我们将描述保存至少 2 年的动脉的无冰冷冻保存后状态。此外，在此目标期间，我们将表征无冰的化学和生物材料特性将使用冷冻宏观镜检测冰来评估冷冻保存的血管的有效玻璃化。在目标 3 中，我们将通过拉曼光谱分析形成和冷冻保护剂残留。两种猪血管模型（股动脉和肺动脉进入颈动脉）的移植研究（28 天）和肺，分别），以验证我们的技术用于未来的 IIb SBIR 提案，使用临床相关的临床前非人类灵长类动物模型和人体组织。