Factors affecting ice formation in cells and their relevance to cryopreservation

Summary

Principal Investigator: Peter Mazur
Affiliation: University of Tennessee
Country: USA
Abstract: DESCRIPTION (provided by applicant): Cryopreservation demands that lethal intracellular freezing (IIF) not occur. There are two routes to its prevention. One is referred to as equilibrium slow cooling. In this procedure, cells are cooled slowly enough so that they lose nearly all their water osmotically before reaching the nucleation temperature at which IIF becomes possible. Many cell types can be easily and successfully cryopreserved by this method, but many others can not, one example being mouse and human oocytes. The second route to avoiding IIF is to subject cells to vitrification procedures, which convert cell water into a glass. To achieve vitrification, the belief is that cells have to be suspended in high concentrations of a permeating CPA and cooled and warmed at high rates, with a reciprocal relation between CPA concentration and rate. This has led us into an investigation in mouse oocytes of the effects of cooling and warming rates and holding temperatures on the growth of intracellular ice by recrystallization during warming. We have found that this lethal process occurs over a range of at least -80[unreadable]C to -50[unreadable]C with a large increase in rapidity with increasing temperature. Companion studies with "vitrified" oocytes indicate that their survival depends almost entirely on the warming rate with cooling rate having relatively little effect. A new specific aim will pursue further studies on this matter. An important question is how does external ice cross the cell membrane to initiate ice nucleation within the cell. A corollary in multi-cellular systems is how does internal ice in one cell propagate to its neighbors? To investigate these questions, we are proposing in this supplemental application to intensify our study of whether two types of pores in two types of cell systems serve as routes for ice transmission. The two types of pores are those in aquaporins and those in gap junctions. The first cell type is the mouse 8-cell/morula embryo. Early 8-cell embryos possess neither aquaporins or gap junctions;late 8-cell (= early morula) embryos possess both. The second cell type are tissue-culture cells;namely, hamster V79 cells and human neuroblastoma cells. By appropriate transfection, both can be obtained with or without functional aquaporins or gap junctions. An important element of the latter work will be the involvement of Dr. David Spray (Einstein College of Medicine) as a collaborator. Dr. Spray is a world authority on gap junctions. In April, 2007, NCRR held a workshop to review the status of the cryopreservation of sperm, oocytes, and embryos of important laboratory research animals, including the zebrafish;however, to date, all attempts to cryopreserve their embryos have failed. For several reasons, immature oocytes may be more amenable. One reason is that their permeability to water and CPA is considerably higher than that of mature oocytes or embryos. Consequently, we propose in a new specific aim to investigate IIF in the immature oocytes. This study will be strongly enhanced by the addition of Dr. Mary Hagedorn (Smithsonian Institution) as a co-investigator. Her expertise is zebrafish cryobiology, an area in which she has made major contributions. PUBLIC HEALTH RELEVANCE (provided by applicant): The ability to preserve cells by freezing to low temperatures has important implications and applications to assisted reproduction and tissue transplantation in medicine, to improving agricultural productivity, to the maintenance of the gemplasm of genetically important laboratory animals far more cost effectively that its maintenance by living colonies, and to the preservation of the germplasm of endangered species.
Funding Period: 2003-08-08 - 2010-08-31
more information: NIH RePORT

Top Publications

  1. pmc Ultra-rapid warming yields high survival of mouse oocytes cooled to -196°c in dilutions of a standard vitrification solution
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee, USA
    PLoS ONE 7:e36058. 2012
  2. pmc Regulatory volume decrease in COS-7 cells at 22 °C and its influence on the Boyle van't Hoff relation and the determination of the osmotically inactive volume
    Diana Peckys
    Fundamental and Applied Cryobiology Group, Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932, USA
    Cryobiology 65:74-8. 2012
  3. pmc The temperature and type of intracellular ice formation in preimplantation mouse embryos as a function of the developmental stage
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee
    Biol Reprod 82:1198-205. 2010
  4. pmc Comparison between the temperatures of intracellular ice formation in fresh mouse oocytes and embryos and those previously subjected to a vitrification procedure
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996 0840, USA
    Cryobiology 61:155-7. 2010
  5. pmc Simple, inexpensive attainment and measurement of very high cooling and warming rates
    F W Kleinhans
    Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, 37932, USA
    Cryobiology 61:231-3. 2010
  6. doi Microfluidic system for transmission electron microscopy
    Elisabeth A Ring
    Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 2215 Garland Ave, Nashville, TN 37232 0615, USA
    Microsc Microanal 16:622-9. 2010
  7. pmc Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification
    Peter Mazur
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996 0840, USA
    Cryobiology 62:1-7. 2011
  8. pmc Stability of mouse oocytes at -80 °C: the role of the recrystallization of intracellular ice
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996 0840, USA
    Reproduction 141:407-15. 2011
  9. pmc Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy
    Diana B Peckys
    Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    Nano Lett 11:1733-8. 2011
  10. pmc Fully hydrated yeast cells imaged with electron microscopy
    Diana B Peckys
    Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    Biophys J 100:2522-9. 2011

Scientific Experts

Detail Information

Publications29

  1. pmc Ultra-rapid warming yields high survival of mouse oocytes cooled to -196°c in dilutions of a standard vitrification solution
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee, USA
    PLoS ONE 7:e36058. 2012
    ..Again, the cooling rate is of less consequence...
  2. pmc Regulatory volume decrease in COS-7 cells at 22 °C and its influence on the Boyle van't Hoff relation and the determination of the osmotically inactive volume
    Diana Peckys
    Fundamental and Applied Cryobiology Group, Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932, USA
    Cryobiology 65:74-8. 2012
    ..48 vs. 0.30). These results indicate the importance of examining cells for volume regulatory mechanisms before performing measurements to determine their osmotic parameters...
  3. pmc The temperature and type of intracellular ice formation in preimplantation mouse embryos as a function of the developmental stage
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee
    Biol Reprod 82:1198-205. 2010
    ..1 +/- 1.5 degrees C. This marked rise in nucleation temperature coincides with the appearance of aquaporin 3 and gap junctions in early morulae (compacted eight-cell), and is presumably causally related...
  4. pmc Comparison between the temperatures of intracellular ice formation in fresh mouse oocytes and embryos and those previously subjected to a vitrification procedure
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996 0840, USA
    Cryobiology 61:155-7. 2010
    ..32 degrees C higher than that for previously vitrified cells. Our conclusion then is that there is no difference between the IIF temperatures of fresh and previously vitrified cells...
  5. pmc Simple, inexpensive attainment and measurement of very high cooling and warming rates
    F W Kleinhans
    Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, 37932, USA
    Cryobiology 61:231-3. 2010
    ..The thermocouple output voltage was read with an inexpensive USB based digital oscilloscope interfaced to a laptop computer, and the raw data were processed with MS Excel...
  6. doi Microfluidic system for transmission electron microscopy
    Elisabeth A Ring
    Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 2215 Garland Ave, Nashville, TN 37232 0615, USA
    Microsc Microanal 16:622-9. 2010
    ..The system allows for rapid (within a minute) liquid exchange, which can potentially be used, for example, to investigate the response of specimens, e.g., eukaryotic or bacterial cells, to certain stimuli...
  7. pmc Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification
    Peter Mazur
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996 0840, USA
    Cryobiology 62:1-7. 2011
    ....
  8. pmc Stability of mouse oocytes at -80 °C: the role of the recrystallization of intracellular ice
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996 0840, USA
    Reproduction 141:407-15. 2011
    ..From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes worldwide in dry ice...
  9. pmc Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy
    Diana B Peckys
    Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    Nano Lett 11:1733-8. 2011
    ..The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles...
  10. pmc Fully hydrated yeast cells imaged with electron microscopy
    Diana B Peckys
    Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    Biophys J 100:2522-9. 2011
    ..Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells...
  11. pmc Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37932 2575, USA
    Reproduction 142:505-15. 2011
    ..AQP3 pores can supposedly be closed by low pH or by treatment with double-stranded Aqp3 RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested...
  12. pmc Rectification of the water permeability in COS-7 cells at 22, 10 and 0°C
    Diana B Peckys
    Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
    PLoS ONE 6:e23643. 2011
    ....
  13. pmc A biologist's view of the relevance of thermodynamics and physical chemistry to cryobiology
    Peter Mazur
    Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, M407 Walters Life Sciences Building, Knoxville, TN 37996 0840, USA
    Cryobiology 60:4-10. 2010
    ..5) The importance of being aware of the assumptions underlying thermodynamic models of cryobiological events. And (6), the difficulties in obtaining experimental verification of thermodynamic and physical-chemical models...
  14. pmc Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope
    Diana B Peckys
    Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, United States of America
    PLoS ONE 4:e8214. 2009
    ..Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory...
  15. pmc The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996 0840, USA
    Cryobiology 59:75-82. 2009
    ..We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization...
  16. pmc Electron microscopy of whole cells in liquid with nanometer resolution
    N de Jonge
    Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232 0615, USA
    Proc Natl Acad Sci U S A 106:2159-64. 2009
    ..The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods...
  17. ncbi Effects of hold time after extracellular ice formation on intracellular freezing of mouse oocytes
    Peter Mazur
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932 2575, USA
    Cryobiology 51:235-9. 2005
    ..For example, in 1M EG, the IIF temperatures are -23.7 and -39.2 degrees C with 0 and 2 min hold times; in 1.5M EG, the corresponding IIF temperatures are -29.1 and -40.8 degrees C...
  18. ncbi Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry
    F W Kleinhans
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932, USA
    Cryobiology 52:128-38. 2006
    ..We conclude that early stage oocytes are a good model system in which to investigate modulators of IIF, but that late stage oocytes are damaged during EIF and infrequently supercool...
  19. ncbi Distinct transport selectivity of two structural subclasses of the nodulin-like intrinsic protein family of plant aquaglyceroporin channels
    Ian S Wallace
    Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996 0840, USA
    Biochemistry 44:16826-34. 2005
    ....
  20. ncbi Extra- and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes
    James F Guenther
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville 37932 2575, USA
    Cryobiology 52:401-16. 2006
    ..Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53]. Perhaps that is a reflection of their much larger size...
  21. pmc Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest
    F W Kleinhans
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932 2575, USA
    Cryobiology 54:212-22. 2007
    ..New experimental EG work will be required to resolve this issue...
  22. pmc The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature
    Peter Mazur
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932 2575, USA
    Cryobiology 54:223-33. 2007
    ..However, the data in toto demonstrate that cell volume is not a determining factor in the IIF temperature...
  23. pmc Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling
    Peter Mazur
    Fundamental and Applied Cryobiology Group, Department of Biochemistry, and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932 2575, USA
    Cryobiology 55:158-66. 2007
    ..We conclude that 30 min at -25 degrees C removes nearly all intracellular freezable water, the consequence of which is that IIF occurs neither during the subsequent rapid cooling to -70 degrees C nor during warming...
  24. pmc Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium--a revisit
    Peter Mazur
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932 2575, USA
    Cryobiology 56:22-7. 2008
    ..With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol...
  25. pmc Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling
    Shinsuke Seki
    Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, 10515 Research Drive, Suite 300 10, Knoxville, TN 37932 2575, USA
    Cryobiology 56:171-80. 2008
    ..A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 degrees C. These findings have significant implications both for cryobiology and cryo-electron microscopy...
  26. pmc Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice
    Shinsuke Seki
    Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37932 2575, USA
    Biol Reprod 79:727-37. 2008
    ..When warmed slowly, they were killed, apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing are discussed...
  27. pmc Intracellular ice formation in yeast cells vs. cooling rate: predictions from modeling vs. experimental observations by differential scanning calorimetry
    Shinsuke Seki
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932 2575, USA
    Cryobiology 58:157-65. 2009
    ..IIF disrupts the plasma membrane; consequently, in a subsequent freeze cycle, the cell can no longer supercool and will not exhibit a second exotherm. This proved to be the case at cooling rates >20 degrees C/min...
  28. pmc Determination of the water permeability (Lp) of mouse oocytes at -25 degrees C and its activation energy at subzero temperatures
    F W Kleinhans
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932 2575, USA
    Cryobiology 58:215-24. 2009
    ..A number of assumptions are required for these water loss calculations and the resulting value of Lp can vary by up to a factor of 2, depending on the choices make...
  29. ncbi Extra- and intracellular ice formation in mouse oocytes
    Peter Mazur
    Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37932 2575, USA
    Cryobiology 51:29-53. 2005
    ..The results are somewhat ambiguous as to which of these characteristics best correlates with IIF...