Mechanisms of freezing damage

Freezing of aqueous systems involves numerous simultaneous changes but this review concentrates on direct effects of the formation of ice and the consequent concentration of solutes in the remaining liquid phase. It is generally believed that cell injury at low cooling rates is principally due to the concentration of both intracellular and extracellular electrolytes and that cryoprotectants act by reducing this build-up. New experimental data are presented to support this explanation; we find that the extent of damage to human red blood cells during freezing in solutions of sodium chloride/glycerol/water can be quantitatively accounted for by the increase in solute concentration. However, we also show that a given degree of damage occurs at lower concentrations of solute in the presence of higher concentrations of glycerol; it appears that glycerol contributes an element of damage itself. Recently published studies from Mazur's laboratory have suggested that the dominant damaging factor at low cooling rates is actually the reduction of the quantity of unfrozen water rather than the corresponding increase in salt concentration that accompanies freezing. These data are re-evaluated, and it is argued that the experimental results could equally well be explained by a susceptibility of cells to shrinkage and re-expansion as the concentration of external impermeant solutes first increases during freezing and then decreases during thawing. It is concluded that external ice probably has no directly damaging effect upon dilute suspensions of cells. However, it is also argued that ice is directly damaging whenever it forms intracellularly, and also when it forms extracellularly in densely packed cell suspensions. In the latter case the damage is probably due to recrystallization of the ice masses during thawing. Extracellular ice also has a directly damaging effect when tissues and organs are frozen. The difficulties of designing experimental methods that will yield unequivocal results is emphasized, and consequently the above conclusions must be regarded as tentative at the present time.