Ice Nucelation in Biological Tissues - Implications to Cryopreservation

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DESCRIPTION (provided by applicant): Summary: The use of liver tissue cells either as isolated hepatocyte suspensions or whole liver tissue slices represents a promising approach to temporary replacement of liver function using bioartificial liver devices. However, the bioartificial liver devices need a readily available source of hepatocytes and whole liver tissue slices, as do the xenobiotic studies of clinical drugs. One technique capable of achieving long term storage of liver cells and tissue slices is the use of low temperatures, i.e. cryopreservation. Although extensive research has been performed to determine the effect of freezing protocol and cryopreservation agents on the viability of hepatocytes and whole liver slices, the development of efficient cryopreservation protocols is still hindered by a lack of fundamental understanding of the physicochemical properties governing the response of whole liver tissue cells to freezing injury. Hypotheses: Knowledge of intracellular ice formation in liver tissue sections will enhance our ability to rationally design and optimize freeze/storage protocols. To test the hypothesis, we will measure the kinetics of ice nucleation in liver tissue sections using a combination of 2 recently developed techniques, one based on low temperature microscopy and another on calorimetry. The proposed technique, if successful, will give the biologist or bioengineer a complete understanding of the freezing processes in tissue sections and will ultimately, lead to better long-term storage protocols for native tissues. Specific Aims: In order to test our hypotheses, the following aims will be accomplished. SA 1: Validation of the proposed technique. Comparison of the ice nucleation data in single isolated cells obtained using the new technique and with a well established cryomicroscopy procedure. SA2: Extension of the technique to measure ice nucleation kinetics in whole tissues. SA3: Modeling and prediction of ice nucleation kinetics in whole tissue sections.