C-2030 : Investigation of anti-freeze proteins effect on vitrification–devitrificartion processes in a micro-scale view

Abstract

Ice crystals can greatly damage biological matter which is kept at low temperature for preservation. This problem can be avoided using vitrification, a process by which a liquid is solidified into a non-crystalline (glassy) state by lowering the temperature in the presence of solutes and greatly increasing the viscosity. This process is also beneficiary since it avoids the formation of concentration gradients introduced during crystallization and thereby avoids osmotic injuries resulting in loss of sample viability. Since the late thirties, when Luyet raised the idea of cells cryopreservation by vitrification, a lot of progress has been made in understanding this process and developing this technique to suit biological needs. However, there is still the main difficulty which has not come into a successful solution – the phenomena called ’devitrification’, when ice crystals form during the rewarming step of partially vitrified biological material. We suggest a natural alternative to face this challenge by using antifreeze proteins (AFPs) found in cold-adapted organisms. These proteins are known to inhibit ice growth in slightly supercooled conditions, and inhibit recrystalization of ice in frozen tissues. In this study we investigate the influence of AFPs on the vitrification and de-vitrification processes under a microscope equipped with controlled cold stage and by differential scanning calorimetry (DSC). Observation on vitrification and de-vitrification of Me2SO solutions at different volumes, temperatures and cooling and warming rates have demonstrated these processes in a micro scale. Preliminary results show that addition of the hyperactive AFP fromTenibrio molitor in various concentrations appear to have less ice nucleation centers during warming. Moreover, ice crystals, at post vitrification temperatures (i.e ∼−105 °C), display slower growth in solutions containing AFPs. These novel effects of TmAFP will be further characterized and developed in a course of utilizing these findings into biological matter cryopreservation applications.