EMBRYO / OOCYTE VITRIFICATION PROTOCOL

Vitrification is an exciting new technique which is used to cryopreserve (freeze) supernumerary eggs and embryos

Vitrification is an exciting new technique which is used to cryopreserve (freeze) supernumerary eggs and embryos. Using ultrarapid (flash) freezing techniques, it's now possible to efficiently store spare eggs and embryos, so they can be used later on to achieve a pregnancy after thawing. Survival rates after vitrification are better than they were with conventional slow freezing.

This describes the laboratory protocol we use for freezing eggs and embryos.

The principle is simple. Vitrification is dependent on the placement of the oocyte/embryo in a very small volume of vitrification medium which is then cooled at an extreme rate, so that the embryos are flash frozen and preserved intact.

MATERIALS REQUIRED

Media : CVM Vitrification Medium (Supplier :COOK IVF)

The vitrification kit (K-SIBV-5000) is a three-step freezing system containing Cryobase buffer with ethylene glycol, DMSO and trehalose as the cryoprotectants.

• Vial 1 Cryobase Buffer.
• Vial 2 Cryobase Buffer with 8% DMSO (v/v) and 8% ethylene glycol (v/v)
• Vial 3 Cryobase Buffer with 16% DMSO (v/v) and 16% ethylene glycol (v/v) and 0.68 M Trehalose.
• Vial 4 DMSO (This solution becomes ice when refrigerated)

1. Falcon 3037 Center-well Dishes
2. Flexipet 170 u
3. Fibreplug
4. Goblets
5. Thermocol Box
6. Liquid nitrogen (LN2)
7. Forceps for handling fibreplug in LN2

Preparation of Vitrification Solution 2 and Vitrification Solution 3

Remove solution 4 from refrigerator and keep it for thawing , as solution 4 contains DMSO, it always becomes ice once refrigerated.

Vitrification Solution 2 = 400ul Solution 4 + 4.6 ml Solution 2

Vitrification Solution 3 = 1 ml Solution 4 + 5.25 ml of Solution 3

FREEZING PROTOCOL

1. Equilibrate Vitrification solution 1, 2 & 3 to 37 C
2. Place them into center-well dishes
3. Label the fibreplugs
4. Immerse the sleeve into LN2 for cooling.
5. Select embryos to be cryopreserved from culture dish and Place them into dish 1, maximum of 6 embryos. Wash them.
6. Set the timer for 3 minutes but do not start the timer.
7. Move all the embryos to Dish 2 for 2 minutes. Start the timer.
8. With one minutes remaining on the timer move the embryos to Dish 3.
9. Complete the next step i.e. loading of embryos onto cryoloop and plunging it into LN2 within 20-25 sec.

Note : loading embryos onto cryoloop is the most difficult part in the procedure. One has to make sure that the volume of media should be as low as possible, since the freezing medium a has high concentration of cryoprotectant and is embryotoxic, which can affect the survival of embryos.

10. Once you plunge the fibreplug into LN2, stir the fibreplug for about 1 minute and without removing it from LN2, put it into the sleeve.
11. Repeat the steps for all set of embryos.
12. Put all the fibreplugs into Goblet and place the goblet into the canister.