Vitrification Assessment: Thermal Analysis of Cryoprotective Aqueous Solutions 1,2 Propanediol and Ethylene Glycol

Abstract

Cryopreservation of viable cells and cell materials is being developed for biological and biopharmaceutical applications. The inhibition of ice formation during the cooling and warming phase of vitrified living biological samples is important for their survival. The tendency to form glasses (glass transition temperature, T_g) upon cooling in the vitrification solution and the stability of the amorphous state upon warming to determine the critical cooling rate (V_ccr) and critical warming rates (V_cwr) are evaluated. The study of thermal properties of ethylene glycol (EG) and 1,2-propanediol (PD) solutions were performed to improve vitrification through better understanding of their molecular mobility and viscosity. Two sets of aqueous solutions were tested. In group A, 35% EG (w/w) was added to different PD concentrations (5%, 10%, and 15%). In group B, 20% PD (w/w) was combined with varying concentrations of EG (20%, 24%, 27%, and 30%). Using the semiempirical model of Boutron, the values of V_ccr and V_cwr for group A were 10, 8, <2.5°C/min, and 1.65 × 10⁵, 678, 32°C/min, respectively. For group B, the values were 24, 10, <2.5, <2.5°C/min, and 9.5 × 10³, 144, 48, 7°C/min, respectively. While the values of V_ccr and V_cwr for 40% EG were 123 and 8.84 × 10⁵°C/min, respectively. The methyl group in PD enhanced the vitreous state, lowering the melting point. Adding a small concentration of PD (3%) to VM3 vitrification solution improved and increased the T_g and enhanced their thermal stability. Analyzing the thermal properties of cryoprotectant is useful when designing the cryopreservation protocols.