[Intracellular free calcium content increase in the erythrocytes treated with the cryoprotective medium based on...

Changes in intracellular free calcium content ([Ca2+]i) in human erythrocytes treated with the cryoprotective medium based on low toxic polymer--polyethylene glycol 1500 (PEG-1500) and then transferred to physiologic salt solution containing 2 mM CaCl2 were studied using fluorescent calcium probe--fura-2. A method of [Ca2+]i calculation with allowance for haemolysis of the cells during the experiment was proposed. It was shown that ignorance of the cell haemolysis resulted in significantly higher [Ca2+]i values obtained. Significant time-dependent increase of [Ca2+]i in the cells treated with PEG-1500 cryoprotective medium at +4 degrees C as well as at +22 degrees C (without freezing) and then transferred in the 2 mM CaCl2 containing physiological salt solution at +37 degrees C was observed. Freezing-thawing of the cells treated with the PEG-1500 cryoprotective medium enhanced haemolysis and further accumulation of calcium in the cells. The results of the study prove that the use of PEG-1500-based cryoprotective medium which does not require washing for human erythrocytes will be accompanied by progressive destruction (haemolysis) of the cells in the blood vessels and may have some negative consequences connected with [Ca2+]i increase in the cryopreserved erythrocytes.