Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling

The formation of ice crystals within cells (IIF) is lethal. The classical approach to avoiding it is to cool cells slowly enough so that nearly all their supercooled freezable water leaves the cell osmotically before they have cooled to a temperature that permits IIF. An alternative approach is to cool the cell rapidly to just above its ice nucleation temperature, and hold it there long enough to permit dehydration. Then, the cell is cooled rapidly to −70 °C or below. This approach, often called interrupted rapid cooling, is the subject of this paper. Mouse oocytes were suspended in 1.5 M ethylene glycol (EG)/PBS, rapidly cooled (50 °C/min) to −25 °C and held for 5, 10, 20, 30, or 40 min before being rapidly cooled (50 °C/min) to −70 °C. In cells held for 5 min, IIF (flashing) occurred abruptly during the second rapid cool. As the holding period was increased to 10 and 20 min, fewer cells flashed during the cooling and more turned black during warming. Finally, when the oocytes were held 30 or 40 min, relatively few flashed during either cooling or warming. Immediately upon thawing, these oocytes were highly shrunken and crenated. However, upon warming to 20 °C, they regained most of their normal volume, shape, and appearance. These oocytes have intact cell membranes, and we refer to them as survivors. We conclude that 30 min at −25 °C removes nearly all intracellular freezable water, the consequence of which is that IIF occurs neither during the subsequent rapid cooling to −70 °C nor during warming.