Rabbit Embryo Vitrification Using Cryogenic Straw: An Ultra-Rapid Cooling Technique to Preserve Rabbit Embryos for Long-term Storage
Transcrição
Embryo vitrification is an ultra-rapid cryopreservation technique that cools embryos at extremely fast rates to ultra-low temperatures, facilitating their long-term storage.
To begin, take harvested rabbit embryos in suitable media. Incubate the embryos with equilibrating solution containing low concentrations of cell-permeating cryoprotectants – ethylene glycol, or EG, and dimethyl sulfoxide, or DMSO. EG and DMSO diffuse through the cell membranes, replacing water molecules inside the cells and preventing intracellular ice-crystal formation.
Next, incubate the embryos with vitrification solution containing higher concentrations of EG and DMSO to enable adequate permeation of the cryoprotectants and sufficient intracellular water efflux, protecting cells from freezing damage.
Couple a sterile cryogenic straw carrier to a microdispenser. Using the microdispenser, fill the straw with desired media to appropriate level. Then, aspirate an air bubble, followed by minimal volume of embryo-containing vitrification solution, and another air bubble.
Aspirate media again until the first media fraction just wets and polymerizes the powder-coated cotton plug, sealing the end of straw. Close the other end with a hydrophobic straw plug. The air and media segments help isolate embryos within the closed carrier.
Briefly plunge the straw into liquid nitrogen at -196°C. The carrier ensures high cooling rates, solidifying embryos into a glass-like amorphous phase, while the low volume of vitrification solution and cryoprotectants used mitigate osmotic stress. The vitrified embryos, with metabolic activity suspended, can be stored until use.
