Embryo vitrification is a rapid cryopreservation technique that allows for the long-term storage of embryos at ultra-low temperatures. This method involves the use of cryoprotectants to prevent ice formation within cells during freezing.
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.
For embryonic vitrification, immerse the embryos in equilibrating solution for 2 minutes, followed by a 1-minute incubation in vitrification solution. At the end of the incubation, load the embryos into a 125-microliter plastic ministraw, and couple the closed end of the straw with an appropriate 1-milliliter microdispenser. Aspirate base medium up to one-third of the straw length, followed by a small air bubble.
Next, use a stereomicroscope to aspirate the embryos in 40 microliters of vitrification solution, followed by another small air bubble in enough base medium to move first liquid fraction up to the cotton end of the ministraw. Then, close the opened end of the ministraw with a straw plug, plunge the ministraw directly into liquid nitrogen to achieve vitrification, and store the ministraw in a liquid nitrogen dewar.
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