Solid CO2 freezing of mouse embryos.

Eight-cell mouse embryos were frozen rapidly to -196 degrees C in the presence of glycerol by a two-step procedure; the lower two-thirds of each tube containing embryos was placed directly from 0 degrees C into crushed solid CO2 for some time before transfer to liquid nitrogen. The cooling rate between -10 and -60 degrees C was approximately 30 degrees C/min. Suitable conditions for the survival of embryos in this fashion were found to be: stepwise addition of glycerol and no seeding; 1 X 8 M-glycerol; 5-30 min equilibration time; 10-30 min holding time in crushed solid CO2; approximately 500 degrees C/min thawing rate. A relatively high proportion (62%) of frozen embryos survived after rapid thawing, but none survived slow thawing. Eight-cell embryos frozen-thawed in this fashion and transferred to recipients developed into normal young.

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