136 The effect of antifreeze proteins on vitrification–devitrificartion processes in a micro-scale view

Abstract

Vitrification, an “ice-free” cryopreservation method, is an effective way to preserve biological matter, as it avoids the damages of ice growth. This method involves solidification of a liquid into a glass state without crystallization due to high values of viscosity at low temperatures. In the recent years it became widely used in preserving oocytes, embryos, organs and seeds. However this method encounters some difficulties in achieving successful preservation. The main difficulty is avoiding ice crystal formation during rewarming, a phenomena called de-vitrification. So far the solutions found to face this problem are: to rewarm at extremely high rates in a homogeneously manner (Mazur, 1992); to use synthetic anti-nucleators, such as polyvinyl alcohol (PVA) (Wowk, 2000); or to use cryoprotectant mixtures at high concentration. The later may be too toxic to maintain sample viability. Solutions with less toxicity that avoid devitrification are desirable. Natural antifreeze proteins (AFPs), found in cold-adapted organisms, might be an alternative for the aforementioned solutions. While interacting with ice crystals, AFPs arrest ice growth at temperatures below the equilibrium melting temperature and also inhibit damaging recrystalization processes. We investigate the influence of AFPs on the vitrification and de-vitrification processes using optical cryo-microscopy. We use the Linkam MDBCS196 controlled cold stage, which can get to liquid nitrogen temperatures with relatively high cooling rates (5000 °C/min). The microscopy includes bright field and fluorescence microscopy with labeled AFPs. Vitrification and de-vitrification of various cryoprotectant solutions as Me 2 SO and Glycerol at different volumes, temperatures and cooling and warming rates were examined. At slow cooling rates in which crystallization occurs, results show that addition of hyperactive AFP from Tenibrio molitor drops the nucleation temperature of the solution and thus extends its suppercooling range. Also, initial results show that while Me 2 SO solution at 30% v/v concentration facilitate vitrification when the sample was cooled at the high rate of the stage, at 20%, addition of AFP at tens of micromolar concentrations were required to achieve vitrification. Devitrification has been observed in both cases and will be further investigated. These results indicate that the AFPs are a promising candidate to reduce the toxicity of cryoprotectant solutions for vitrification. Source of funding: The European Research Council (ERC). Conflict of interest: None declared. ido.braslavsky@mail.huji.ac.il