C-1002: Survivals approach 100% for mouse oocytes suspended in one-third concentration of vitrification media, vitrified, and warmed ultra-rapidly by an IR laser pulse

Abstract

Vitrification is the most sought after route to the cryopreservation of animal oocytes and embryos and other cells of medical, genetic, and agricultural importance. Standard vitrification has been based on two firmly held premises. One is that the vitrification solution in which the cells are suspended must have a very high concentration of a mixture of non-electrolytic solutes, some of which can permeate the cell and some of which cannot. We have used EAFS10/10, a solution developed by Pedro et al., where E, A, F, and S refer to ethylene glycol (EG), acetamide, Ficoll, and sucrose. The total molality is 7.38 molal, of which 6.5 molal is permeating (EG and acetamide), and the remainder are non-permeating. The second premise is that avoiding ice formation in cells and obtaining high survivals demands the highest of cooling rates. Consequently, a series of devices have been developed over the past decades that achieve cooling rates of ⩾10,000 °C/min by using very small volumes of cell suspensions. One example is the Cryotop. So, current thinking is that successful vitrification requires that cells be suspended in high concentrations of protective solutes and that they be cooled at very high rates to below -100°C. We report here that neither of these beliefs hold for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultrarapid rates (10,000,000 °C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only one third of the standard. Full paper published on-line 3/22/14 at http://dx.doi.org/10.1016/j.cryobiol.2014.02.005 . Research supported by NIH grant R01OD011201 .