Instability of Frozen Human Erythrocytes at Elevated Temperatures

Abstract

Frozen cells are known to be unstable at elevated subzero temperatures; however, the kinetics of cell damage as a function of storage temperature and time are not well understood. The present study investigated the instability of frozen human erythrocytes during isothermal storage at elevated subzero temperatures. The relationship between the instability of frozen cells and the temperature-dependent state/phase transitions in frozen domains was examined. Human erythrocytes were cryopreserved with 12% (wt/vol) hydroxyethyl starch in phosphate-buffered saline solution by plunging into liquid nitrogen, and were then isothermally stored at elevated subzero temperatures. Hemolysis following thawing and dilution was used as an indicator of cell damage during isothermal storage. The instability of frozen cells was found to conform to a special form of the Johnson–Mehl–Avrami model, H(T, t) = H(T)[1 - exp( -kt)], where H(T, t) represented the percent hemolysis at temperature T after time t, H(T) was the maximal hemolysis value, and k was the rate constant. Values of the equation parameters as a function of temperature were derived. Calorimetric analysis revealed a complex thermal transition profile upon warming of the frozen sample. These transitions resulted from the formation of heterogeneous domains during freezing and further phase separation (concentration) due to devitrification (crystallization) during warming. However, the instability of frozen cells at elevated subzero temperatures may not be attributed to the temperature-dependent state/phase transitions in frozen domains alone. The relevance of frozen cell instability to successful freeze-drying is discussed.