Cryomicroscopic and Calorimetric Assessment of Cell Response During Freezing Process

Abstract

Understanding the biophysical processes that govern freezing responds of cells is an important step in characterizing and improving the cryopreservation. The quantitative analysis on cell volume shrinkage during freezing helps us understand the mechanism of cryopreservation. Freezing studies were conducted using a Linkam cryostage fitted to an optical microscope cooled under controlled rates at 5, 10, 20, 50 and 100°C/min. The volume of renal cell at subzero temperature have been quantified by heat latent obtained from DSC data and compared to the microscopic data. Experimental data were fitted by nonlinear regression method to calculate the water transport parameters. Also, the final volume of cell was predicted. The ice crystal formation is the vital factor for cryopreservation. Cooling rate deeply affected ice formation temperature. The faster cells are cooled, the more their contents supercool, and at some subzero temperature that supercooled cytoplasm will freeze. Intracellular ice formation (IIF) plays a central role in cell injury during cooling. Cryomicroscope and differential scanning calorimeter were used to study the relationship between cooling rate and TEIF and TIIF. And morphological changes of renal cell were also obtained by cryomicroscope. According to cryomicroscope and DSC experiments, IIF did not occur in renal cells cooled at ≤10°C/min. High cooling rate could depress the ice formation temperature.