Type of Document	Master's Thesis
Author	Acharya, Tathagata
Author's Email Address	tachar1@lsu.edu
URN	etd-11072008-013716
Title	Freezing Processes in Cell Suspensions Evaluated Using Cryomicroscopy
Degree	Master of Science in Mechanical Engineering (M.S.M.E.)
Department	Mechanical Engineering
Advisory Committee	Advisor Name Title Devireddy, Ram Committee Chair Wong, Harris Committee Member Monroe, William Todd Committee Member
Keywords	cryomicroscopy intracellular ice formation water transport
Date of Defense	2008-10-14
Availability	unrestricted
Abstract This thesis aims at evaluating the freezing response of three different cell types, Pacific Oyster embryos, Jurkats and Helas, using the technique of cryomicroscopy. The choice of cells was primarily based on supporting ongoing research work at the Bioengineering Laboratory, Department of Mechanical Engineering at Louisiana State University in Baton Rouge. On a secondary basis, the cells were chosen based on their contrasting nature. While Pacific Oyster being a favorite food in USA calls for successful techniques of cryopreservation of their embryos in order to keep up with the growing demand, Jurkat and HeLa are undesired malignant human cells that require successful cryosurgical techniques for their destruction. The fourth chapter of the thesis addresses the freezing experiments performed on Oyster embryos at freezing rates of 5 deg C/min and 10 deg C/min. During these experiments, embryos were investigated for either dehydration (water transport) or intracellular ice formation (IIF). The next two chapters address the freezing experiments performed on Jurkat cells and HeLa cells respectively. Freezing rates ranging from 1 deg C/min to 50 deg C/min were used for these cells. Once dehydration was observed, the cells were examined for their volume shrinkage. A graph of temperature against normalized volume was plotted using the experimental results. The key cell level parameters were: Reference permeability of cell membrane to water (Lpg), apparent activation energy (ELp), inactive cell volume (Vb), and the ratio of surface area for water transport to the volume of intracellular water (SA/WV). The values of ‘Vb’ for the chosen cells were known from earlier literature. The experimental data was fit into the water transport equation, using a numerical model, in order to obtain the values of the unknown cell level parameters i.e. Lpg and ELp. Finally, Generic Optimal Cooling Rate Equation (GOCRE) was used to determine the optimal cooling rate for the chosen variety of cells. Hence, higher freezing rates were used on the cells, which were investigated for IIF. IIF observed using cryomicroscopy, through darkening probably supported the results for optimal freezing rates, obtained using the water transport experiments and subsequent numerical simulations.
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