Factors affecting ice formation in cells and their relevance to cryopreservation

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DESCRIPTION (provided by applicant): Intracellular ice formation (IIF) is usually lethal. The main concern of the first four years of this grant was to determine details of IIF in oocytes of mice and Xenopus and in yeast. The details included the temperatures at which IIF occurred and the effect of cryoprotectant (CPA) type and concentration on those temperatures. From these studies, we have found that in 1 to 1.5 M EG or glycerol, nucleation occurs near -400C, the homogeneous nucleation temperature of water. But in lower concentration, it occurs by heterogeneous nucleation, with a high probability that external ice is the nucleator. (See papers 139-140, 142-143, 145-147, 149-150, 153 by Mazur, Seki, and Kleinhans in the Progress Report Publication List). In the past three years under the current renewal grant, we have shifted the emphasis to determining physical events during vitrification and recrystallization of internal ice, and their effects on survival. The most important finding has been that under certain conditions, the formation of intracellular ice is not lethal; what is lethal is the growth of those crystals during warming by recrystallization. This translates to the fact that the warming rate plays the dominant role in determining survival of "vitrified" mouse oocytes-and not the cooling rate. (Papers 157, 154, and 160). The current renewal application builds directly on the items in the above paragraph. Up to now, we have based "survivals" on the retention of osmotic properties by thawed cells and by the morphological normality of cell membranes and cytoplasm. The argument was that those sets of conditions that yielded low "survivals" based on these criteria, would almost certainly exhibit very low or zero functional survival. We found, however, that a number of conditions yielded osmotic/morphological survivals of 80 to 90% if the warming rates were very high; the cooling rate had less or no effect. Now we intend to determine the functional survival of oocytes treated in this way. Another finding has been that if the oocytes are warmed > 100,0000C/min, some 80% survive osmotically, morphologically, and in their ability to develop to 2-cell embryos even if the vitrification medium is diluted in half. Possibly, even faster warming would permit the use of even more dilute vitrifying solutions. We propose to test this hypothesis by using a special laser to achieve warming rates that are 100- times higher. Most believe that the efficacy of vitrification solutions depends on the very high solute concentrations in them. Another possibility is that their efficacy depends more on the degree to which they osmotically dehydrate the cells prior to initiating cooling to -1960C, We propose to test that hypothesis in a quantitative manner.