Cryomicroscopic Investigations of Freezing Processes in Cell Suspensions

Abstract

This study evaluates the freezing response of three different cell types, Pacific oyster embryos (~50 µm in diameter), Jurkat cells and HeLa cells (~12 to 15 µm’s in diameter), using cryomicroscopy. Freezing experiments were performed on oyster embryos at cooling rates of either 5 or 10 °C/min, while Jurkats were cooled at either 1 or 10 °C/min and HeLa cells were cooled at either 1, 15 or 20 °C/min, respectively. The experiments with oyster embryos were primarily designed to investigate the phenomena of intracellular ice formation (IIF) while the experiments for Jurkat and HeLa cells were designed to investigate both cellular dehydration and IIF during freezing. IIF was characterized by the abrupt black flashing during the cooling steps while the cellular dehydration experiments were characterized by the volumetric (projected area) shrinkage of the cells during the cooling steps. Mathematical models were fit to the cellular dehydration data to obtain the Jurkat and HeLa cell membrane permeability (Lpg) at the reference temperature (273.15 K), the apparent activation energy of the cellular dehydration process (ELp) or the temperature dependence of Lpg. The temperature dependence of IIF in the oyster embryos, the Jurkat cells and the HeLa cells were also determined.