C-1015: Implications of the consequences of laser-induced ultra-rapid warming of mouse oocytes and embryos to vitrification theory and to the successful vitrification of other cell types

Abstract

Jin, Kleinhans, and Mazur have reported in this meeting that up to 100% of oocytes and 2-cell embryos survive vitrification in a 3-fold dilution of EAFS medium if warmed at 10,000,000 °C/min by an IR laser pulse. The usual cooling rate was 69,000 °C/min, but 95% also survived when cooled at 10,000 °C/min. Thus, survival is far more dependent on the rate of warming than on the rate of cooling, the reason being that very high warming rates prevent or impede the recrystallization of intracellular ice. In all our experiments, the oocytes or embryos were held for 2.0 min in 1/3 × EAFS before vitrification That time was chosen because Pedro (1997) found it to produce the best survival after vitrification. EAFS media are strongly hyperosmotic so that oocytes/embryos placed in them, undergo an osmotic shrink-swell pattern with time. There is an initial rapid shrinkage to a minimum volume due to water loss, followed by a much slower re-swelling due to the slow influx of the permeating solutes. Based on Pedro’s data, that minimum occurs near 2 min, and osmotic theory argues that at that point, the cells have lost 85% of their original water and very little permeating solute has entered them. The strong inference, then, is that the high subsequent survivals are a consequence of the 85% osmotic dehydration and are not due to the intracellular presence of permeating solutes. Supporting that conclusion is Jin and Mazur’s report that 77% of oocytes and 95% of 2-cell and 8-cell embryos survive vitrification when suspended in media containing only the non-permeating solutes sucrose provided they are warmed at 1 × 107 °C/min by a pulse from our IR laser. None survived when warmed 100× more slowly in the absence of the laser. These findings are at odds with orthodox beliefs about the causes of injury from vitrification. They could possibly open the way to the successful vitrification of important cell types that to date have been partially or fully refractory to this approach.