Isochoric vitrification: An experimental study to establish proof of concept

Ice-free vitreous cryopreservation (vitrification) is regarded as the principal method for avoiding ice crystallization damage in cryopreserved tissues and organs. We previously established the fundamental thermodynamics of isochoric (constant volume) systems for cryopreservation, and now extend this novel approach to vitrification in an isochoric system. This was achieved by measuring pressure changes in a 2 ml isochoric chamber containing a variety of aqueous solutions of the ubiquitous cryoprotective additives (CPA), dimethyl sulfoxide (Me₂SO) and Propane-diol. The CPAs, ranging in concentrations from 0 to 49%(w/v), were prepared in a proprietary preservation solution (Unisol^®) in anticipation of future applications to tissue and organ banking. Pressures developed in the system were monitored as a function of CPA concentration and cooling rate when the isochoric chamber was cooled to cryogenic temperature (-160 °C). This study corroborated our previous findings that pressure increases in accordance with the thermodynamics of partially frozen systems of low concentrations of CPAs. A key finding of this study was that in an isochoric system of higher concentrations of CPA, which vitrifies, there is no increase in pressure. In fact, an increase in pressure is a measure of failure to vitrify and a measure of devitrification. Comparison with results from the literature show that the concentration of CPAs needed for vitrification in an isochoric chamber is substantially lower than that needed for vitrification in isobaric systems at 1 atm and hyperbaric systems at 1000 atm. In addition, isochoric chambers are much more effective in promoting vitrification than hyperbaric pressure chambers, and are less expensive, easier to design, and implement.