Injury to human granulocytes at low temperatures

Granulocytes differ from other blood cells in that they are more sensitive to injury on freezing and thawing. Previous studies suggest that the difficulty in preserving them is related to their sensitivity to osmotic stress. A miniaturized system both for freezing granulocytes and testing their function in the same Terasaki plates has been developed. This allowed study of several factors simultaneously including concentration of protective additive, different cooling conditions, and dilution conditions on rewarming.We observed two types of injury to granulocytes frozen to higher subzero temperatures and thawed directly. The first type was initially severe but decreased with time in the frozen state under some conditions and appears not to have been reported in other cell systems. The second type of injury consists of conventional loss of function with longer holding times after freezing. Cells surviving these two classes of injury could be protected against the further stress of rapid cooling into liquid nitrogen, but this protection required a longer time during cooling in the frozen state than with other cell types.We have studied the interactions between several variables, e.g., time in DMSO before freezing and dilution rate after thawing in an attempt to characterize the unusual injurious mechanism at high subzero temperatures that, we believe, is the real cause of the difficulty of preserving these cells.