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. 1993 Dec;65(6):2524–2536. doi: 10.1016/S0006-3495(93)81319-5

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

J O Karlsson 1, E G Cravalho 1, I H Borel Rinkes 1, R G Tompkins 1, M L Yarmush 1, M Toner 1
PMCID: PMC1225995  PMID: 8312489

Abstract

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.

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Selected References

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

  1. Borel Rinkes I. H., Toner M., Ezzell R. M., Tompkins R. G., Yarmush M. L. Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration. Cryobiology. 1992 Aug;29(4):443–453. doi: 10.1016/0011-2240(92)90047-6. [DOI] [PubMed] [Google Scholar]
  2. Borel Rinkes I. H., Toner M., Sheeha S. J., Tompkins R. G., Yarmush M. L. Long-term functional recovery of hepatocytes after cryopreservation in a three-dimensional culture configuration. Cell Transplant. 1992;1(4):281–292. doi: 10.1177/096368979200100405. [DOI] [PubMed] [Google Scholar]
  3. Chesné C., Guillouzo A. Cryopreservation of isolated rat hepatocytes: a critical evaluation of freezing and thawing conditions. Cryobiology. 1988 Aug;25(4):323–330. doi: 10.1016/0011-2240(88)90040-5. [DOI] [PubMed] [Google Scholar]
  4. Fahy G. M., MacFarlane D. R., Angell C. A., Meryman H. T. Vitrification as an approach to cryopreservation. Cryobiology. 1984 Aug;21(4):407–426. doi: 10.1016/0011-2240(84)90079-8. [DOI] [PubMed] [Google Scholar]
  5. Fuller B. J., Lewin J., Sage L. Ultrastructural assessment of cryopreserved hepatocytes after prolonged ectopic transplantation. Transplantation. 1983 Jan;35(1):15–18. doi: 10.1097/00007890-198301000-00004. [DOI] [PubMed] [Google Scholar]
  6. Gómez-Lechón M. J., Lopez P., Castell J. V. Biochemical functionality and recovery of hepatocytes after deep freezing storage. In Vitro. 1984 Nov;20(11):826–832. doi: 10.1007/BF02619627. [DOI] [PubMed] [Google Scholar]
  7. Hubel A., Toner M., Cravalho E. G., Yarmush M. L., Tompkins R. G. Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel. Biotechnol Prog. 1991 Nov-Dec;7(6):554–559. doi: 10.1021/bp00012a011. [DOI] [PubMed] [Google Scholar]
  8. Innes G. K., Fuller B. J., Hobbs K. E. Functional testing of hepatocytes following their recovery from cryopreservation. Cryobiology. 1988 Feb;25(1):23–30. doi: 10.1016/0011-2240(88)90016-8. [DOI] [PubMed] [Google Scholar]
  9. Li R., Trounson A. Rapid freezing of the mouse blastocyst: effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen. Reprod Fertil Dev. 1991;3(2):175–183. doi: 10.1071/rd9910175. [DOI] [PubMed] [Google Scholar]
  10. MAZUR P. KINETICS OF WATER LOSS FROM CELLS AT SUBZERO TEMPERATURES AND THE LIKELIHOOD OF INTRACELLULAR FREEZING. J Gen Physiol. 1963 Nov;47:347–369. doi: 10.1085/jgp.47.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mazur P. Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. Cell Biophys. 1990 Aug;17(1):53–92. doi: 10.1007/BF02989804. [DOI] [PubMed] [Google Scholar]
  12. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol. 1984 Sep;247(3 Pt 1):C125–C142. doi: 10.1152/ajpcell.1984.247.3.C125. [DOI] [PubMed] [Google Scholar]
  13. McCaa C., Diller K. R., Aggarwal S. J., Takahashi T. Cryomicroscopic determination of the membrane osmotic properties of human monocytes at subfreezing temperatures. Cryobiology. 1991 Aug;28(4):391–399. doi: 10.1016/0011-2240(91)90046-q. [DOI] [PubMed] [Google Scholar]
  14. Novicki D. L., Irons G. P., Strom S. C., Jirtle R., Michalopoulos G. Cryopreservation of isolated rat hepatocytes. In Vitro. 1982 Apr;18(4):393–399. doi: 10.1007/BF02796340. [DOI] [PubMed] [Google Scholar]
  15. Rall W. F., Mazur P., McGrath J. J. Depression of the ice-nucleation temperature of rapidly cooled mouse embryos by glycerol and dimethyl sulfoxide. Biophys J. 1983 Jan;41(1):1–12. doi: 10.1016/S0006-3495(83)84399-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rall W. F., Reid D. S., Farrant J. Innocuous biological freezing during warming. Nature. 1980 Jul 31;286(5772):511–514. doi: 10.1038/286511a0. [DOI] [PubMed] [Google Scholar]
  17. Rijntjes P. J., Moshage H. J., Van Gemert P. J., De Waal R., Yap S. H. Cryopreservation of adult human hepatocytes. The influence of deep freezing storage on the viability, cell seeding, survival, fine structures and albumin synthesis in primary cultures. J Hepatol. 1986;3(1):7–18. doi: 10.1016/s0168-8278(86)80140-4. [DOI] [PubMed] [Google Scholar]
  18. Toner M., Cravalho E. G., Stachecki J., Fitzgerald T., Tompkins R. G., Yarmush M. L., Armant D. R. Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential. Biophys J. 1993 Jun;64(6):1908–1921. doi: 10.1016/S0006-3495(93)81562-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yarmush M. L., Toner M., Dunn J. C., Rotem A., Hubel A., Tompkins R. G. Hepatic tissue engineering. Development of critical technologies. Ann N Y Acad Sci. 1992 Oct 13;665:238–252. doi: 10.1111/j.1749-6632.1992.tb42588.x. [DOI] [PubMed] [Google Scholar]

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