NONEQUILIBRIUM FREEZING OF ONE-CELL MOUSE EMBRYOS - MEMBRANE INTEGRITY AND DEVELOPMENT POTENTIAL

A thermodynamic model was used to evaluate and optimize a rapid three- step rapid three-step nonequilibrium freezing protocol for one-cell mo use embryos in the absence of cryoprotectants (CPAs) that avoided leth al intracellular ice formation (IIF). Biophysical parameters of one-ce ll mouse embryos were determined at subzero temperatures using cryomic roscopic investigations (i.e., the water permeability of the plasma me mbrane, its temperature dependence, and the parameters for heterogeneo us IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120-degrees-C/min wh en a 5-min holding period was inserted at -10-degrees-C to assure cell ular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conven tional embryo cryopreservation cooling rates of between 0.5 and 1-degr ees-C/min. At slow cooling rates, embryos predominantly follow the equ ilibrium phase diagram and do not undergo IIF, but mechanisms other th at IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our therm odynamic model using a programmable freezer and confirmed the theoreti cal predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% aft er freezing down to -45-degrees-C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as asses sed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the develo pmental competence of the embryos.