Analysis of cryopreservation media thermophysical characteristics after ultra-rapid cooling through differential scanning calorimetry

Cryoprotective agents play a critical role in minimizing cell damage caused by ice formation during cryopreservation. However, high concentrations of CPAs are toxic to cells and tissues. Required concentrations of CPAs can be reduced by utilizing higher cooling and warming rates, but insight into the thermophysical properties of biological solutions in the vitrification method is necessary for the development of cryopreservation protocols. Most studies on thermophysical properties under ultra-rapid cooling conditions have been qualitatively based on visualization. Differential scanning calorimetry methods are ideal for studying the behavior of biomaterials in various freezing conditions quantitatively and accurately, though previous studies have been predominantly restricted to slower cooling rates. Here, we developed an ultra-rapid cooling method for DSC that can achieve minimal cooling rates exceeding 2000 °C/min. We investigated the thermophysical vitrification behavior of ternary solutions of phosphate buffer saline (1X), dimethyl sulfoxide or glycerol and ice blocking polymers (X-1000 or Z-1000). We quantified the impact of solute concentration on ice crystal formation during rapid cooling. Our findings support the expectation that increasing the solute concentration reduces the amount of ice formation, including devitrification. Devitrification increases from 0 % to 40 % (v/v) Me₂SO and then reduces significantly. The relative amounts of devitrification to the total ice formation are 0 %, 60 %, 0 % in 20 %, 40 %, 60 % (v/v) Me₂SO, and 2 %, 48 %, 49 % in 20 %, 40 %, 60 % (v/v) glycerol, respectively. The results suggest that at low concentrations, such as below 20 % (v/v) for Me₂SO or glycerol, increasing the warming rate after ultra-rapid freezing is not essential to eliminate devitrification. Furthermore, ice blocking polymers do not reduce ice formation substantially and cannot eliminate devitrification under ultra-rapid cooling conditions. In conclusion, our results provide insights into the impact of solute concentration on ice formation and devitrification during rapid cooling, which can be practical for optimizing cryopreservation protocols.