Osmotic effects of dilution on erythrocytes after freezing and thawing in glycerol-containing buffer

Red blood cells frozen in 1.7 M and particularly in 2.2 M of glycerol retain a high degree of integrity upon thawing as long as the dilution procedure of the cryoprotectant is slow and preferentially compensated by the addition of sorbitol. As the nonpenetrating cryoprotectant sorbitol induces initial cell shrinkage, cell swelling upon dilution of the cryoprotectants may not lead to hemolysis. However, rapid dilution of glycerol even with buffer containing up to 0.50 M of sorbitol cannot be achieved without provoking considerable hemolysis. Due to the relative slow rate at which glycerol leaves the cells, membrane damage to the younger cell populations remains considerable and is even more pronounced in the older cell groups. The dramatic osmotic changes occurring during the dilution process lead to the formation of aberrant cell populations as demonstrated by the red cell size frequency distribution curves.