Developing a device for visualization of large-scale cryopreservation

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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.