Validation of a simple and effective sterile vitrification system for embryos

Abstract

OBJECTIVE: In a decade of escalated concern and awareness for good tissue handling practices (GTPs) and FDA regulations, cryopreservation methods that emphasize secure storage, technical ease/repeatability, and consistent outcomes are needed. DESIGN: Frozen-thawed 1-cell mouse embryos (Embryo-Tech) were used to validate a novel double container vitrification procedure. MATERIALS AND METHODS: The vitrified (VTF) embryos were pipette into shortened (cut: 1.5-2.0 cm) denuding pipettes (275-300 μm ID), detached, loaded tip-end first and then LN2 plunged /stored in sealed CBS™ High Security straws. Mouse 2-cell and blastocyst (BL) stage embryos (5/straw) were VTF and rewarmed using Irvine Scientific kits. A preliminary expt. (I) was performed to test BL survivability compared to conventional freezing. In expt.2, 2-cell embryos were VTF and cultured to the BL stage. In expt. 3, cooling and warming rate curves were validated using micro-thermocouples and a datalogging device. In expt. 4, BL survivability was reassessed using a modified warming step where the tip was immediately placed in warm H-HTF solution for 10 sec upon removal and then reattached to a Stripper™ device to decant embryos and pipette them serially. All embryos were cultured in LG medium + 5% HSA in 25μl microdroplets. RESULTS: In expt. 1, 67% re-expansion of re-frozen BL's occurred, similar to controls. In expt. 2, 16 to 20 VTF 2-cells produced ≥ expanded BLs (80%), similar to control cultured 2-cells (80% BL). In expt. 3, cooling and warming rates inside the CBS straws adjacent to, as well as inside, the contained VS-stripper tip itself were measured, warming in methanol or in air. The VTF-tip sealed in a CBS™ straw cooled at a rate of 1000 ± 100°C/ min between −10°C and −140°C. Warming rates were similar with both methods up to −70°C, but slowed beyond that in air. To better maintain high warming rates above −70°C, we placed the isolated tip in warm H-HTF for 10 sec before securing the tip for embryo removal. In expt. 4, 25 full to hatched mouse BLs were VTF and rewarmed using the latter method, with all 25 BLs recovered, surviving and developing at 24 hr (100%), being similar to LAH treated controls and VS-exposed BLs. CONCLUSIONS: The data support the promising effectiveness of a novel VTF procedure which offers safe, sterile LN2 storage and technical simplicity, with excellent recovery and survival rates. Additional studies with different solutions are needed to optimize the safety of this procedure for vitrifying human oocytes and embryos.