54 Cryoprotectant and cooling-rate dependence of ice formation in bovine oocytes during cooling and warming probed by time-resolved X-ray diffraction

In vitrification-based oocyte cryopreservation, post-thaw survival depends on the amount of damage incurred during cooling and warming, which depends in part on the amount, type, and grain size of ice formed. Ice formed during cooling depends on cryoprotective agent (CPA) concentration and cooling rate. Ice formed during warming also depends on ice formed during cooling and on the warming rate. Time-resolved X-ray diffraction is used to study how cooling rates, warming rates, and CPA concentration affect ice formation in bovine oocytes during both cooling and warming. Our hypothesis is that increasing cooling and warming rates will allow reduction of CPA concentrations required to achieve acceptable maximum ice fractions and maximum ice crystal sizes during the freeze-thaw cycle. Bovine MII oocytes from slaughterhouse ovaries were cooled under various conditions (n = 350, 10 oocytes per cooling speed and CPA concentration) at cooling rates between ∼2,000°C min⁻¹ and >600,000°C min⁻¹ using a NANUQ automated cryocooler for crystallography; with oocytes held on different supports (Cryotops [Kitazato Corporation] and ultra-low-thermal mass MicroLoops LD); soaked with a standard CPA solution (15% dimethyl sulfoxide [DMSO], 15% ethylene glycol [EG]) at strengths between 100% and 40%, with constant (0.5 M) or decreasing sucrose concentration; and with liquid on the oocyte surface replaced by oil (to verify that observed ice diffraction originated from inside the oocyte). X-ray diffraction and optical images were recorded from oocytes at 100 K and then as oocytes were warmed at ∼60,000°C min⁻¹ by solenoid-controlled high speed gas stream, at the Cornell High-Energy Synchrotron Source. Ice-to-cell volume ratio was compared between cooling groups using a mixed model ANOVA in SAS v9.4. Cryocooled oocytes showed no ice, small grain cubic ice, small grain stacking-disordered ice, or larger grain hexagonal ice, depending on cooling rate, sample support, and CPA concentration. On warming, all samples—including initially ice-free samples—developed ice, progressing from cubic to stacking-disordered, to hexagonal ice before melting. Ice grain sizes at higher CPA concentrations always remained modest, but larger grains developed when using lower CPA concentrations and slower cooling and warming rates. Thermal response times of oocytes on Cryotops were 3–5 times larger than on MicroLoops LD. Using fully optimised cooling protocols (cooling rate of >600,000°C min⁻¹ using MicroLoops LD), oocytes could be reliably vitrified with either no visible ice diffraction or only faint and diffuse cubic diffraction using CPA concentrations down to and including 6% DMSO, 6% EG, and 0.2 M sucrose, and developed only small grain ice on warming. Time-resolved X-ray diffraction allows direct and detailed characterisation of ice formation in oocytes during cooling and, more importantly, during warming. Improved cooling technologies allow large (∼60%) reduction in CPA concentrations required to adequately inhibit ice formation.