Glucose and glycerol temperature-pressure correlations for the design of cryopreservation protocols in an isochoric system at subfreezing temperature.

There is growing interest in the use of isochoric (constant volume) freezing for cryopreservation of biological matter. The goal of this study is to generate fundamental experimental data on the pressure temperature relation during the freezing of an isochoric system of aqueous solutions of two compounds, glucose and glycerol. Glucose and glycerol are commonly used as cryoprotectants in conventional isobaric (constant pressure) cryopreservation protocols. Earlier studies have shown that the increase in pressure during isochoric freezing is detrimental to biological matter and limits the range of temperatures in which isochoric freezing can be used for preservation to temperatures corresponding to pressures below 40 MPa. In physiological saline solution this pressure corresponds to a temperature of - 4 °C. Our new experimental data shows that the addition of 2 M glycerol to the saline solution lowers the temperature at which the isochoric freezing pressure is 40 MPa to -11 °C, 3 M glycerol to - 16.5 °C, and 4 M glycerol to - 24.5 °C, thereby substantially expending the range of temperatures in which cryopreservation by isochoric freezing can be practiced.