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

Abstract

Crioprotectors are the main component of any vitrification media. The penetrant crioprotectors are essential for cell dehydration and for the decrease of the freezing point of the solution, allowing a longer time for dehydration to set in. The aim of our paper was to make a series of experiments in order to determine the concentration at which four cryoprotectants (ethylene glycol, sucrose, Ficoll 70 and galactose) singly and in pairs would vitrify on plunging into liquid nitrogen and remain vitreous when thawed in water bath. A total of 156 solutions were tested. During freezing, vitrification was evidenced by the formation of transparent glass when the unsealed straws were plunged into liquid nitrogen, at -196°C. Crystallization (ice formation) resulted in a milky appearance. Solutions that vitrify on freezing were tested if they remain vitreous on thawing. For thawing we tested three temperatures 20°C, 25°C and 37°C. During thawing, solutions that did not devitrified were transformed from solid clear state to the liquid state without evidence of a milky appearance. From the combinations of two cryoprotectors were tested a number of 51 solutions vitrify on freezing (19 solutions with ethylene glycol and galactose; 19 solutions with ethylene glycol and sucrose; 13 solutions with ethylene glycol and Ficoll). The ethylene glycol and galacose pair give the best results on thawing (3 combinations remained vitreous on thawing) at 37°C.

Keywords

• crioconservation
• vitrification
• ethylene glycol
• galactose
• sucrose
• ficoll70