Temperature dependence of the survival of human erythrocytes frozen slowly in various concentrations of glycerol

Abstract

One widely accepted explanation of injury from slow freezing is that damage results when the concentration of electrolyte reaches a critical level in partly frozen solutions during freezing. We have conducted experiments on human red cells to further test this hypothesis. Cells were suspended in phosphate-buffered saline containing 0-3 M glycerol, held for 30 min at 20 degrees C to permit solute permeation, and frozen at 0.5 or 1.7 degrees C/min to various temperatures between -2 and -100 degrees C. Upon reaching the desired minimum temperature, the samples were warmed at rates ranging from 1 to 550 degrees C/min and the percent hemolysis was determined. The results for a cooling rate of 1.7 degrees C/min indicate the following: (a) Between 0.5 and 1.85 M glycerol, the temperature yielding 50% hemolysis (LT50) drops slowly from -18 to -35 degrees C. (b) The LT50's over this range of concentrations are relatively independent of warming rate. (c) With glycerol concentrations of 1.95 and 2.0 M, the LT50 drops abruptly to -60 degrees C and to below -100 degrees C, respectively, and becomes dependent on warming rate. The LT50 is lower with slow warming at 1 degree C/min than with rapid. With still higher concentrations (2.5 and 3.0 M), there is no LT50, i.e., more than 50% of the cells survive freezing to-100 degrees C. Results for cooling at 0.5 degrees C/min in 2 M glycerol were similar except that the LT50s were some 10-20 degrees C higher. A companion paper (Rall et al., Biophys. J. 23:101-120, 1978) examines the relation between survival and the concentrations of salts produced during freezing.

Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations

“…Higher post-thaw integrities observed at temperatures above 230uC is attributed to the rapid cooling being initiated at higher sub-zero temperatures thus creating an overall faster cooling rate. These results are consistent with those reported elsewhere in the literature using fast cooling rates in conjunction with lower concentrations of glycerol 50,54,55 . When 110 mM b-PMP-Glc is added to RBCs in 15% glycerol, post-thaw RBC integrities are 5-15% better at all sub-zero cooling temperatures higher than 230uC (red solid lines, Fig.…”

Accelerated electrons and microwave irradiation effects on the human erythrocyte membranes

“…During simultaneous and subsequent microwave and energy irradiation, the samples remained in frozen state. The temperature variations in the frozen state before, during and after irradiation did not affect the erythrocyte membrane properties [2] under our experimental conditions (glycerol concentration >2M) [4].…”

Effect of microwaves, high energy electrons and gamma irradiation on the osmotic resistance of human erythrocyte membranes

“…We obtained an improvement in the membrane stability at a specific irradiation time, which expresses itself in a smaller degree of haemolysis. The samples were frozen at -196 ~ before microwave irradiation because the temperature variations in the frozen state of the cryoprotected erythrocytes do not affect the membrane properties [9]. It can be assumed that heating induced in the frozen erythrocytes does not influence the haemolysis rate, and the non-thermal component (that induces specific effects distinct from the changes induced by heating) of electromagnetic radiation [7] has a significant contribution to the results.…”