studies regarding the crioprotective proprieties of the vitrification media, with glycerol, sucrose and ficoll 70 used in embryo cryopreservation

The aim of our paper was to make a series of experiments in order to determine theconcentration at which three cryoprotectants (glycerol, sucrose and Ficoll 70) singlyand in pairs would vitrify on plunging into liquid nitrogen and remain vitreous whenthawed in water bath. As penetrating cryoprotector we used glycerol (MW=92.09,Farmachim) and as un-penetrating cryoprotectors we used sucrose (MW=342.3,Sigma S7903) and Ficoll 70 (MW= 60,000-80,000, Sigma F4375). For glycerolthere were tested concentrations from 1M to 6.5M, with concentrations step of 0.5M.For the non penetrating cryoprotectors there were tested concentrations of 5%, 10%,15% and 20%. There were a total number of 116 solutions tested. The solutionvitrification ability on freezing was tested by plunging directly into liquid nitrogen at-196°C. There were tested three thawing temperatures 20°C, 25°C and 37°C. Theconcentration at which glycerol solution passed into vitreous state was 5M, but atthawing none of them remained vitreous at thawing. When pairs of cryoprotectorswere tested 44 solutions vitrified at freezing (23 for glycerol-sucrose, 21 forglycerol-Ficoll 70) and 11 of them remained vitreous at thawing. The glycerol-Ficoll70 pair give the best results on thawing (7 solution remained vitreous on warming)at 37°C.