STUDIES REGARDING THE CRIOPROTECTIVE PROPRIETIES OF THE VITRIFICATION MEDIA, WITH DMSO, SUCROSE, FICOLL 70 AND GALACTOSE USED IN EMBRYO CRYOPRESERVATION

Abstract

The aim of our paper was to make a series of experiments in order to determine the concentration at which four cryoprotectants (DMSO, sucrose, Ficoll 70 and galactose) singly and in pairs would vitrify on plunging into liquid nitrogen and remain vitreous when thawed in water bath. As penetrating cryoprotector we used DMSO (MW=78.13 Da, Sigma D5879) and as nonpenetrating cryoprotectors we used sucrose (MW=342.3 Da, Sigma S7903), Ficoll 70 (MW= 60,000-80,000 Da, Sigma F4375) and Galactose (MW = 180,16 Da; Sigma G 6152). For DMSO there were tested concentrations from 1M to 6.5M, with concentrations step of 0.5M. For the nonpenetrating cryoprotectors there were tested concentrations of 5%, 10%, 15% and 20%. There were a total number of 168 solutions tested. The solutions vitrification ability on freezing was tested by direct plunging into liquid nitrogen at - 196°C. Three thawing temperatures were tested 20°C, 25°C and 37°C. The concentration at which DMSO solutions passed into vitreous state was 5M, but at thawing none of them remained vitreous at thawing. When pairs of cryoprotectors were tested 67 solutions vitrified at freezing (23 for DMSO-sucrose, 23 for DMSOFicoll 70 and 21 for DMSO-galactose) and 26 of them remained vitreous at thawing. The DMSO and galacose pair give the best results on thawing (11 solution remained vitreous on warming) at 37°C.

Keywords

• crioprotectors
• vitrification
• dmso
• sucrose
• ficoll 70
• galactose