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Procedure for Warming of Vitrified Embryos

 Freezing Medium and Culture Medium for Mouse Reproductive Engineering

  FERTIUP & CARDMEDIUM

   Reproductive Engineering Manuals

Materials and Equipment

1. Microscope

2. 0.25 M sucrose (Cat#CSR-R-Y077, CSR-R-Y078)

3. KSOM/AA

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

5. Transfer pipettes

6. Micropipette

Procedure

Preparation for Warming

1. Put 3 drops (100µL/drop) of KSOM/AA into a dish and cover them with liquid paraffin. Place the dish in an incubator (37°C, 5% CO2 in air) for at least 30 minutes.

2. Warm 0.25 Msucrose in an incubator (37°C, 5% CO2 in air) before use.

Recovering Vitrified Embryos

1. Remove the required sample from the liquid nitrogen and open the cryotube cap. Discard any liquid nitrogen in the tube and allow it to stand at room temperature for 30 seconds.

2. Add 0.9mL of 0.25M sucrose (preheated to 37°C) to the cryotube and warm the sample quickly via pipetting. When pipetting, take care not to generate large amounts of bubbles and to not physically damage embryos by pipetting too quickly. Once warmed, transfer the contents of the cryotube into a plastic dish.

3. Place 0.4-0.5mL of 0.25M sucrose into the cryotube, and transfer the contents into the plastic dish. This further dilutes the cryoprotectant and ensures that all of the embryos have been transferred.

Note: It is very important to warm the sample quickly to avoid damaging the embryos due to the toxicity of the cryoprotective solution (DAP213).

4. Aspirate the embryos from the liquid and carefully transfer them into a drop of KSOM/AA (washing dish), then keep them in an incubator (37°C, 5% CO2 in air).

5. After 10 minutes, wash the embryos with 2 cycles of fresh KSOM/AA (washing dish).

References

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.

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