Developing a device for visualization of large-scale cryopreservation

描述(申请人提供)：在极低的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的替代方案。虽然单细胞和小组织结构的冷冻保存技术已经成熟，但大型组织和器官的冷冻保存技术仍处于发展阶段。最有前景的大规模冷冻保存技术之一是玻璃化，即抑制冰的结晶，使生物材料处于类玻璃状态(玻璃体在拉丁语中的意思是玻璃)。玻璃化是通过在组织中引入冷冻保护剂(CPA)来实现的，当足够快地冷却时，这种保护剂可以防止冰晶的形成。虽然防冰是成功玻璃化冷冻保存的关键，但玻璃化所需的高冷却速度会导致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)，其最显著的结果是形成骨折。任何低温保存过程的结果不能仅仅在终点--储存和室温--在标本达到平衡时，在冷却或复温阶段发生的物理事件的证据可能已经消失的地方进行评估。一种持续监测冷冻保存过程并记录这种证据的手段代表着一种未得到满足的需求。这项研究建议开发一种新的设备，用于可视化与大样本低温保存过程中的结晶、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、广泛用于低温生物学应用的冷冻机集成在一起。拟议装置的三个主要用途是：(1)作为基本研究工具，探索与低温保存有关的物理影响；(2)作为研究和开发工具，开发新的低温保存方案；(3)作为质量保证工具，与大量生产低温保存产品相结合。扫描冷冻显微镜的开发有四个具体目标：(1)开发放置在现有可控速率冷冻室中的样品的扫描设备；(2)开发集成视频记录、配准和温度数据的软件；(3)演示集成系统在结晶和玻璃化过程中的应用；以及(4)将偏振光集成到扫描设备中并开发相关方法。偏振光的应用已被证明可以增强冰核的识别，以及一种研究玻璃和其他固体透明介质中机械应力发展的手段。 公共卫生相关性(由申请人提供)：组织和器官在极低温度下保存--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的替代方案。这项研究建议开发一种新的物理效应可视化设备，如冰结晶和断裂形成，用于大样本的低温保存应用。防止冰结晶和破裂的形成是低温保存成功的关键。

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. PUBLIC HEALTH RELEVANCE (provided by the applicant): Preservation of tissues and organs at very low temperatures-cryopreservation-appears to be only feasible alternative for providing high quality biomaterial for transplantation medicine. This research proposal concerns the development of a new device for visualization of physical effects, such as ice crystallization and fracture formation, for the application of cryopreservation of large specimens. Preventing ice crystallization and fracture formation is critical for cryopreservation success.