Developing a device for visualization of large-scale cryopreservation

描述（由申请人提供）：在极低温度下保存组织和器官-冷冻保存-似乎是为移植医学提供高质量生物材料的唯一可行选择。虽然单细胞和小组织结构的低温保存技术已经建立，但大型组织和器官的低温保存技术仍处于发展阶段。大规模低温保存最有前途的技术之一被称为玻璃化，其中冰结晶被抑制，生物材料被困在玻璃状状态（玻璃体在拉丁语中的意思是玻璃状的）。玻璃化是通过向组织中引入冷冻保护剂（cpa）来实现的，当冷却速度足够快时，保护剂可以防止冰晶的形成。虽然防冰是玻璃化冷冻成功的关键，但玻璃化所需的高冷却速度会带来另一种潜在的破坏性影响，即热机械应力（由材料随温度下降而收缩的趋势所驱动），其最显著的结果是裂缝的形成。任何低温保存过程的结果都不能仅仅在终点——储存温度和室温——进行评估，在那里，在冷却或再加热阶段发生的物理事件的证据可能在标本达到平衡时已经消失。一种持续监测冷冻保存过程并记录这些证据的方法代表了一种未满足的需求。这项研究计划涉及开发一种新的设备，用于在大型标本冷冻保存过程中可视化与结晶、玻璃化和断裂形成相关的物理效应。新设备概念设计的创新之处在于，它将作为一个附加单元，与商业上使用的、广泛可用的冷冻装置集成在一起，用于低温生物学的应用。设想该设备的三个主要用途：(i)作为基础研究工具，探索与低温保存相关的物理效应；（ii）作为研发工具，开发新的低温保存协议；（iii）作为质量保证工具，与冷冻产品的批量生产相结合。扫描低温显微镜的开发有四个具体目标：(1)开发一种扫描装置，用于放置在可控速率冷冻室中的样品；(2)开发录像、登记、温度数据集成软件；(3)展示集成系统在结晶和玻璃化工艺中的应用；(4)将偏振光集成到扫描装置中并开发相关方法。偏振光的应用已被证明可以增强对冰核的识别，也是研究玻璃和其他固体透明介质中机械应力发展的一种手段。

DESCRIPTION (provided by applicant): Preservation of tissues and organs at very low temperatures-cryopreservation-appears to be only feasible alternative to providing high quality biomaterial for transplantation medicine. While techniques for cryopreservation of single cells and small tissue structures are well established, cryopreservation techniques for bulky tissues and organs are still at the developmental stage. One of the most promising techniques for large-scale cryopreservation is known as vitrification, where ice crystallization is suppressed, and the biological material is trapped in a glassy-like state (vitreous in Latin means glassy). Vitrification is achieved by introducing cryoprotective agents (CPAs) into the tissue, which prevent the formation of ice crystals when cooled rapidly enough. While ice prevention is the key for successful cryopreservation by vitrification, the high cooling rate required for vitrification drives another potentially devastating effect, which is thermo-mechanical stress (driven by the tendency of the material to contract with the decreasing temperature), with fracture formation as its most dramatic outcome. The outcome of any cryopreservation process cannot merely be evaluated at the end points-the storage and room temperatures-where evidence of physical events that took place during the cooling or rewarming stages may have disappeared by the time that the specimen has reached equilibrium. A means to continuously monitor the process of cryopreservation and document such evidence represents an unmet need. This research proposal concerns the development of a new device for visualization of physical effects associated with crystallization, vitrification, and fracture formation during cryopreservation of large specimens. The innovation in the conceptual design of the new device is that it will be integrated-as an add-on unit-with commercially used and widely available freezer for cryobiology applications. Three principal usages are envisioned for the proposed device: (i) as a basic research tool, to explore physical effects associated with cryopreservation; (ii) as a research and development tool, to develop new cryopreservation protocols; and (iii) as a quality assurance tool, to be incorporated with mass production of cryopreserved products. Four specific aims have been set for the development of the scanning cryomacroscope: (1) to develop a scanning device for a specimen placed in the chamber of an available controlled-rate freezer; (2) to develop software for integration of video recording, registration, and temperature data; (3) to demonstrate the application of the integrated system on crystallization and vitrification processes; and, (4) to integrate polarized light into the scanning device and to develop related methodology. The application of polarized light has been proven to enhance identification of ice nucleation, as well as a means to investigate the development of mechanical stress in glasses and other solid transparent media.