Direct concentration measurements of the unfrozen portion of solutions under freezing

Phase diagrams of solutions consisting of cryoprotective agents (CPA) are very useful in cryobiology research. Those diagrams depict the points of solution concentrations at corresponding temperatures: one of essential inputs that can be utilized to compute the volume response of cell under freezing process. However, generating such plots is costly and time-consuming. A direct method is proposed in this study to determine the solution concentration of unfrozen parts at multiple sub-zero temperatures. Measurements of binary solutions, composed of water and sodium chloride, were performed and compared with published data. Ternary solutions, consisting of water, sodium chloride and dimethyl sulfoxide, were also measured. The uniqueness and advantage achieved through the usage of this method are demonstrated when phase diagrams of complex cryopreservation solutions (CryoStor solutions including CryoStor Base and CryoStor 10) are generated. The temperature range where the method is utilized is either limited by the osmometry (0-3200 mmol/kg) or by the availability of liquid samples at sub-freezing temperatures. Modified methods will be required to address the limitation of osmolality measurements and the availability of sub-freezing liquid samples at lower temperatures.