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. 1978 Jul;23(1):89–100. doi: 10.1016/S0006-3495(78)85435-6

Temperature dependence of the survival of human erythrocytes frozen slowly in various concentrations of glycerol.

H Souzu, P Mazur
PMCID: PMC1473548  PMID: 667309

Abstract

One widely accepted explanation of injury from slow freezing is that damage results when the concentration of electrolyte reaches a critical level in partly frozen solutions during freezing. We have conducted experiments on human red cells to further test this hypothesis. Cells were suspended in phosphate-buffered saline containing 0-3 M glycerol, held for 30 min at 20 degrees C to permit solute permeation, and frozen at 0.5 or 1.7 degrees C/min to various temperatures between -2 and -100 degrees C. Upon reaching the desired minimum temperature, the samples were warmed at rates ranging from 1 to 550 degrees C/min and the percent hemolysis was determined. The results for a cooling rate of 1.7 degrees C/min indicate the following: (a) Between 0.5 and 1.85 M glycerol, the temperature yielding 50% hemolysis (LT50) drops slowly from -18 to -35 degrees C. (b) The LT50's over this range of concentrations are relatively independent of warming rate. (c) With glycerol concentrations of 1.95 and 2.0 M, the LT50 drops abruptly to -60 degrees C and to below -100 degrees C, respectively, and becomes dependent on warming rate. The LT50 is lower with slow warming at 1 degree C/min than with rapid. With still higher concentrations (2.5 and 3.0 M), there is no LT50, i.e., more than 50% of the cells survive freezing to-100 degrees C. Results for cooling at 0.5 degrees C/min in 2 M glycerol were similar except that the LT50s were some 10-20 degrees C higher. A companion paper (Rall et al., Biophys. J. 23:101-120, 1978) examines the relation between survival and the concentrations of salts produced during freezing.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bank H., Mazur P. Visualization of freezing damage. J Cell Biol. 1973 Jun;57(3):729–742. doi: 10.1083/jcb.57.3.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bank H. Visualization of freezing damage. II. Structural alterations during warming. Cryobiology. 1973 Jun;10(2):157–170. doi: 10.1016/0011-2240(73)90023-0. [DOI] [PubMed] [Google Scholar]
  3. Diller K. R. Intracellular freezing: effect of extracellular supercooling. Cryobiology. 1975 Oct;12(5):480–485. doi: 10.1016/0011-2240(75)90029-2. [DOI] [PubMed] [Google Scholar]
  4. LOVELOCK J. E. The haemolysis of human red blood-cells by freezing and thawing. Biochim Biophys Acta. 1953 Mar;10(3):414–426. doi: 10.1016/0006-3002(53)90273-x. [DOI] [PubMed] [Google Scholar]
  5. LOVELOCK J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim Biophys Acta. 1953 May;11(1):28–36. doi: 10.1016/0006-3002(53)90005-5. [DOI] [PubMed] [Google Scholar]
  6. Mazur P., Leibo S. P., Miller R. H. Permeability of the bovine red cell to glycerol in hyperosmotic solutions at various temperatures. J Membr Biol. 1974;15(2):107–136. doi: 10.1007/BF01870084. [DOI] [PubMed] [Google Scholar]
  7. Mazur P., Miller R. H. Permeability of the human erythrocyte to glycerol in 1 and 2 M solutions at 0 or 20 degrees C. Cryobiology. 1976 Oct;13(5):507–522. doi: 10.1016/0011-2240(76)90144-9. [DOI] [PubMed] [Google Scholar]
  8. Mazur P., Miller R. H. Survival of frozen-thawed human red cells as a function of the permeation of glycerol and sucrose. Cryobiology. 1976 Oct;13(5):523–536. doi: 10.1016/0011-2240(76)90145-0. [DOI] [PubMed] [Google Scholar]
  9. Mazur P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology. 1977 Jun;14(3):251–272. doi: 10.1016/0011-2240(77)90175-4. [DOI] [PubMed] [Google Scholar]
  10. McGrath J. J., Cravalho E. G., Huggins C. E. An experimental comparison of intracellular ice formation and freeze-thaw survival of HeLa S-3 cells. Cryobiology. 1975 Dec;12(6):540–550. doi: 10.1016/0011-2240(75)90048-6. [DOI] [PubMed] [Google Scholar]
  11. Miller R. H., Mazur P. Survival of frozen-thawed human red cells as a function of cooling and warming velocities. Cryobiology. 1976 Aug;13(4):404–414. doi: 10.1016/0011-2240(76)90096-1. [DOI] [PubMed] [Google Scholar]
  12. Morris G. J., Farrant J. Interactions of cooling rate and protective additive on the survival of washed human erythrocytes frozen to -196 degrees C. Cryobiology. 1972 Jun;9(3):173–181. doi: 10.1016/0011-2240(72)90029-6. [DOI] [PubMed] [Google Scholar]
  13. RAPATZ G., LUYET B. Electron microscope study of erythrocytes in rapidly frozen frog's blood. Biodynamica. 1961 Aug;8:295–315. [PubMed] [Google Scholar]
  14. Rall W. F., Mazur P., Souzu H. Physical-chemical basis of the protection of slowly frozen human erythrocytes by glycerol. Biophys J. 1978 Jul;23(1):101–120. doi: 10.1016/S0006-3495(78)85436-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rapatz G., Luyet B. Combined effects of freezing rates and of various protective agents on the preservation of human erythrocytes. Cryobiology. 1968 Mar-Apr;4(5):215–222. doi: 10.1016/s0011-2240(68)80001-x. [DOI] [PubMed] [Google Scholar]
  16. Rapatz G., Luyet B., MacKenzie A. Effect of cooling and rewarming rates on glycerolated human erythrocytes. Cryobiology. 1975 Aug;12(4):293–308. doi: 10.1016/0011-2240(75)90003-6. [DOI] [PubMed] [Google Scholar]
  17. Rapatz G., Sullivan J. J., Luyet B. Preservation of erythrocytes in blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature. Cryobiology. 1968 Jul-Aug;5(1):18–25. doi: 10.1016/s0011-2240(68)80139-7. [DOI] [PubMed] [Google Scholar]
  18. Shepard M. L., Goldston C. S., Cocks F. H. The H2O-NaCl-glycerol phase diagram and its application in cryobiology. Cryobiology. 1976 Feb;13(1):9–23. doi: 10.1016/0011-2240(76)90154-1. [DOI] [PubMed] [Google Scholar]

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