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Simple Vitrification of Mouse Embryos

 Freezing Medium and Culture Medium for Mouse Reproductive Engineering

  FERTIUP & CARDMEDIUM

   Reproductive Engineering Manuals

Materials and equipment

1. 1M DMSO (Cat. CSR-R-T072)

2. DAP213 (Cat.# CSR-R-T073)

3. Plastic dish (35mm X 10mm Cat.No.430588; CORNING)

4. Filter unit (Millex-GV 0.22µm Cat.No.SLGV013SL; MILLIPORE)

5. Gel loading tip (MBP Gel 200, Cat.No.3621; Molecular BioProducts)

6. Transfer pipettes

7. Cryotubes (Cryogenic Vials Cat.No.MS-4501W; Sumitomo Bakelite, Japan is recommended. If you cannot get it, use 366656; NUNC.)

8. Micropipette

9. Vial canes

10. Nalgene Labtop Cooler (5115-0012; NALGENE, USA)

11. Liquid nitrogen

12. Microscope

13. 0.25 M sucrose

14. KSOM/AA

15. Liquid paraffin

Procedure

Preparation of a Block Cooler and Cryotubes

1. A day before use, place a block cooler in a freezer at -20°C.

2. About 10 minutes before commencing the vitrifying procedure, take the block cooler out of the freezer.

3. Stand some cryotubes in the block cooler (5115-0012; NALGENE, USA).
About 40 embryos / cryotube are easy to handle; in other words, when you want to vitrify 120 embryos, you need to stand 3 cryotubes in the block cooler.

4. Just before starting the procedure, check the temperature inside of the tubes is at 0°C.]

Vitrification

1. Filter the 1M DMSO and put 4 drops of it (~100µL / drop) into a dish. One drop is to wash the embryos taken from the collection medium, while the others are to hold the washed embryos.

2. Place a group of embryos into one of the 4 drops to rinse them of the collection medium.

Divide the rinsed embryos equally between the other drops.

These aliquots will eventually be transferred to a storage vial.

For example, if one were to collect 120 embryos and vitrify them in 40-embryo aliquots, the embryos would first be placed together in the rinse drop and then divided equally among the three drops.

3. Using a 20µL pipette and a gel-loading tip, transfer the embryos contained within 5µL of 1M DMSO solution into a cryotube.

Once transferred, put the cryotube into the block cooler at 0°C and wait for 5 minutes.

Note: It is possible to keep the cryotubes in the block cooler at 0°C for longer than 5 minutes (<20 minutes).

Note: If the embryos are pushed together in the center of the drop, it is easy to suck them all up in 5µL of the 1M DMSO solution.

4. Add 45µL of cryoprotective solution (DAP213) at 0°C into the cryotube and equilibrate for 5 minutes in the 0°C block cooler.

Note: Do not fasten the caps too tightly after adding the DAP213, or they will be too difficult to remove quickly when samples are recovered from the freezer.

5. Quickly set the cryotubes on a cane and plunge the samples directly into liquid nitrogen.

    Continues to Procedure for Warming of Vitrified Embryos 

References

1. Nakagata N. 1989. High survival rate of unfertilized mouse oocytes after vitrification. J. Reprod. Fert. 87: 479-483.

2. Nakagata N. 1993. Production of normal young following transfer of mouse embryos obtained by in vitro fertilization between cryopreserved gametes. J. Reprod. Fert. 99: 77-80.

3. Nakagata N. 1995. Studies on cryopreservation of embryos and gametes in mice. Exp. Anim. 44: 1-8.

4. Nakao K., Nakagata N., and Katsuki M. 1997. Simple and effcient procedure for cryopreservation of mouse embryos by simple vitrification. Exp. Anim. 46: 231-234. 118

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.