Procedure for Warming of Vitrified Embryos

Materials and Equipment

1. Microscope
2. 0.25 M sucrose
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℃, 5% CO2 in air) for at least 30 minutes.
   
   
2. Warm 0.25 Msucrose in an incubator (37℃, 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℃) 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.
   
   
   [Recovering Vitrified Embryos]
   
   
   Note: It is very important to warm the sample quickly to avoid damaging the embryos due to the toxicity of the cryoprotective solution (DAP213).
   
   
   
   
   
   [Pipetting to Recover Vitrified Embryos]
   
   
   
   
   
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℃, 5% CO2 in air).
   
   
   
   [Micrograph: Embryos Recovered after Vitrification]
   
   
   
   
5. After 10 minutes , wash the embryos with 2 cycles of fresh KSOM/AA (washing dish).
   
   
   
   
   

References

1. Nakao K., Nakagata N., and Katsuki M. 1997. Simple and effcient vitrification procedure for cryopreservation of mouse embryos. Exp. Anim. 46(3): 231-234.

Update history

1. Updated : 11 May, 2011
2. Modified : 6 October, 2012
   The sentence just under the step No.5 of Recovering Vitrified Embryos ("For blastocycts, wash the embryos after 60 minutes.") was deleted, as it was confirmed that keeping blastocysts in the drop for 10 minutes is no problem, just as for 2-cell stage embryos.
3. Modified : 22 January, 2014
   The page title has been changed.
   The old one : Procedure for Thawing of Frozen Embryos.
   The new one : Procedure for Warming of Vitrified Embryos.