Fast Freezing Processes in Tissues

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PROJECT SUMMARY (ORIGINAL) The goal of the research is to advance the formulation of cryopreservation protocols for biological systems using fundamental biophysical parameters. At present, crucial gaps exist in our knowledge of the biophysical parameters governing the states of intra- and extra-cellular water during a fast freezing process. This proposal aims to redress this crucial gap by introducing two new and effective techniques: (a) a measurement technique based on microscale thermoelectric sensors, engineered using microfabrication techniques suitable for use with artificial tissues and (b) a measurement technique based on a combination of calorimetry and low temperature microscopy suitable for use with native tissues. Specifically, the project will: (1) design fabricate and test microscale thermoelectric sensors to measure fast freezing processes in isolated cells (Neonatal Human Dermal fibroblasts, NHDFs) and artificial tissues (NHDFs embedded in a collagen gel sheet) with and without Cryoprotective Agents (CPAs) and (2) develop a combination of calorimetric and low temperature microscopy (directional solidification) methods to assess fast freezing processes in NHDFs and native (mammalian liver) tissue sections with and without CPAs. CPA addition and removal in both tissue (artificial and native) systems will be modeled using irreversible thermodynamic models. These integrated methods from disparate disciplines will lead to rational design and development of freezing processes for cryopreservation of donated tissues, an extremely scarce resource, as well as artificially engineered tissues. The education and infrastructure development part of this NIH AREA 15 proposal includes both instructional and research components for LSU undergraduate students. PROJECT SUMMARY (NIGMS Supplemental Award) The NIGMS supplemental (equipment) award requests funds to purchase a state of the art differential scanning calorimeter (DSC 8500) to enhance the quality of the data obtained in Aim 2 of the project. The new instrument will allow us to limit measurement errors and to assess the true contribution of cell heterogeneity. In addition, the new instrument will controllably freeze cells to a much higher rates (~750 ˚C/min) thus allowing us to fully accomplish the goals of the original proposal, i.e., to assess fast freezing processes in tissues.