Critical to cryopreservation is that lethal intracellular freezing (IIF) not occur. Its occurrence depends on two major factors. One is the cooling rate. It has to be low enough so that the cells lose nearly all their water osmotically before cooling to the temperature at which IIF becomes possible. Second is the temperature at which IIF occurs. The higher that temperature, the greater the difficulty in avoiding IIF by slow cooling. Our current grant and this renewal proposal deal primarily with the latter. IIF in mouse and Xenopus oocytes and in Arabidopsis protoplasts was found to require the presence of extracellular ice in close contact with the cell membrane. One strong piece of evidence in the mouse oocyte is that IIF occurs at temperatures where about 95 percent of the external medium has frozen. That temperature varies from -14?C to -40?C depending on the concentration of cryoprotective compounds in the medium. Another factor affecting the IIF temperature may be cell size, for IIF occurs at much higher temperatures in 1 mm Xenopus eggs than it does in <0.1 mm mouse oocytes. The two tools used above were a cryostage that permits us to observe cells during cooling and warming and manifests IIF as """"""""flashing"""""""", and a differential scanning calorimeter (DSC) that detects IIF as an outburst of heat. In this renewal application we propose to introduce two new instruments. One, in collaboration with the Oak Ridge National Laboratory, is a Scanning and Transmission Electron Microscope (STEM) equipped with a newly designed sample chamber that will permit high resolution images of hydrated cells in aqueous solutions. The other instrument is a directional freezing stage. It permits a cooling rate to be resolved into the thermal gradient (G) and the crystal growth velocity (V). Differences in G and V affect the ice crystal morphology which in turn may affect the temperature of IIF. We will add three new cell types to the study: Yeast and two types of hamster tissue culture cells. These cells are 10-times smaller than mouse oocytes thus permitting us to further explore the role of cell size. Second, cells have to be that small to fit in the STEM liquid sample chamber. Third, IIF has never been observed directly in these cells and we intend to use DSC for this purpose. Other major aims in this renewal proposal are (1) to determine whether the high correlation between observed IIF temperature in mouse oocytes and the frozen fraction holds for other cell types. (2) To determine whether there is a relation between pores in the plasma membrane and the IIF temperature. For this, we will study the freezing of mouse morulae, the 8 to 12 cells of which possess gap junctions and aquaporin pores. Second, we will introduce pores in mouse oocytes and plant protoplasts by exposing them to a toxin from Staphylococcus bacteria. The final goal will be to use the mechanistic data on IIF to devise better methods of avoiding it and thus improve success in the cryopreservation of cells that currently can not be well cryopreserved. ? ? ?

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
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National Center for Research Resources Initial Review Group (RIRG)
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Mirochnitchenko, Oleg
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University of Tennessee Knoxville
Schools of Arts and Sciences
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Seki, Shinsuke; Mazur, Peter (2012) Ultra-rapid warming yields high survival of mouse oocytes cooled to -196°c in dilutions of a standard vitrification solution. PLoS One 7:e36058
Peckys, Diana; Mazur, Peter (2012) 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. Cryobiology 65:74-8
Peckys, Diana B; Kleinhans, F W; Mazur, Peter (2011) Rectification of the water permeability in COS-7 cells at 22, 10 and 0ýýC. PLoS One 6:e23643
Seki, Shinsuke; Mazur, Peter (2011) Stability of mouse oocytes at -80?°C: the role of the recrystallization of intracellular ice. Reproduction 141:407-15
Mazur, Peter; Seki, Shinsuke (2011) Survival of mouse oocytes after being cooled in a vitrification solution to -196ýýC at 95ýý to 70,000ýýC/min and warmed at 610ýý to 118,000ýýC/min: A new paradigm for cryopreservation by vitrification. Cryobiology 62:1-7
Seki, Shinsuke; Edashige, Keisuke; Wada, Sakiko et al. (2011) 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. Reproduction 142:505-15
Peckys, Diana B; Mazur, Peter; Gould, Kathleen L et al. (2011) Fully hydrated yeast cells imaged with electron microscopy. Biophys J 100:2522-9
Peckys, Diana B; de Jonge, Niels (2011) Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy. Nano Lett 11:1733-8
Seki, Shinsuke; Mazur, Peter (2010) Comparison between the temperatures of intracellular ice formation in fresh mouse oocytes and embryos and those previously subjected to a vitrification procedure. Cryobiology 61:155-7
Mazur, Peter (2010) A biologist's view of the relevance of thermodynamics and physical chemistry to cryobiology. Cryobiology 60:4-10

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