Authors
Rahul Thorat and Arvind Ingle
Summary
This protocol describe the cryopreservation of 8-cell to morula-stage mouse embryos using a “slow freezing and fast revival method. An average of twenty five embryos were filled in each 0.25ml capacity plastic straw and embryos were cooled down slowly using a controlled-rate freezing assembly. They were cooled from room temperature (24°C) to -7ºC at the rate of 2ºC/min. by the controlled rate freezer where initial holding time was maintained for 5 min. Manual seeding was done at -7ºC with the help of cotton bud dipped in liquid nitrogen and then temperature of straws were dropped to -30ºC at the rate of 0.5ºC/min and from -30ºC to -120ºC at the rate of 1ºC/min. After holding at -120ºC for 5 minutes, the straws were directly plunged in to the liquid nitrogen for long term storage. After fast revival, viability percentage of embryos was ranging from 51 to 81%. These embryos were kept for 24hrs of incubation in CO2 incubator at 37°C with 5% CO2 where an average of 89% blastocyst formation rate was observed. The developed blastocysts were used for surgical transfer in pseudopregnant female mice to get live births for the re-establishment of the colony. By using this protocol, we can freeze down 8-cell to morula stage embryos of any mouse strain and revive them successfully whenever need arise.Further details
The protocol was published on Protocol Exchange on 14 December 2012. To see the entire protocol, click on the source link.Advertisement
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