Analysis of the Nonequilibrium Phase Change Behaviors of the Cryoprotectant Solutions for Cryopreservation of Human Red Blood Cells with Low-Concentration Glycerol

Recently, we proposed a low-glycerol cryoprotectant formulation (consisting of 0.4 M trehalose and 5% glycerol) for cryopreservation of human red blood cells (RBCs), which greatly reduced the concentration of glycerol, minimized intracellular ice damage, and achieved high recovery. Although this study was successful in cellular experiments, the nonequilibrium phase transition behaviors of the cryoprotective agent solution have not been systematically analyzed. Therefore, it is essential to provide reliable thermodynamic data to substantiate the viability of this cryopreservation technique. In this study, the phase change behaviors and thermal properties of typical trehalose and/or glycerol solutions quenched in liquid nitrogen were investigated using differential scanning calorimetry and cryomicroscopy. It was found that the glass transition temperatures of both the trehalose aqueous solution (<1.0 M) and glycerol aqueous solution (<40% w/v) did not vary apparently with the concentration at low concentrations, while they increased significantly with increasing concentration at high concentrations. Moreover, it was revealed that the inhibitory effect of trehalose on ice growth was affected by glycerol. We further found that the addition of low concentrations of glycerol facilitates the partial glass transition of trehalose solutions at low concentrations. The results of this work provide reliable thermodynamic data to support the cryopreservation of human RBCs with unusually low concentrations of glycerol.