Development of mouse embryos cryopreserved by an ultra-rapid method of freezing.
High concentrations of cryoprotectant combined with sucrose were utilized in an ultra-rapid freezing protocol for mouse preimplantation embryos. Dimethylsulphoxide (DMSO, 1.5 or 3.5 M) or propanediol (PROH, 1.5 or 3.0 M) combined with 0.25 M sucrose were used as freezing solutions. One-, 2- or 8-cell embryos were placed directly into these solutions at room temperature, loaded into straws and plunged into liquid nitrogen within 2-3 min. The straws were rapidly thawed and the embryos expelled into the solution in which they were frozen for 10 min. The cryoprotectants were then removed by single- or multi-step dilution. Survival and development of the embryos in vitro and in vivo were assessed. DMSO (1.5 M) and both concentrations of PROH were totally inadequate as a cryoprotectant in this freezing protocol. A concentration of 3.5 M DMSO gave high survival and development rates when a multi-step dilution procedure was used, but not with a single-step dilution. One-cell embryos gave 71% survival, 35% in-vitro development and 10% in-vivo viability; 2-cell embryos showed 87% survival, 77% in-vitro development and 66% in-vivo viability; and 8-cell embryos showed 97% survival, 87% in-vitro development and 62% in-vivo viability. The results for the 2- and 8-cell stages compared favourably with non-frozen controls, which had 71% in-vivo viability. This method of cryopreservation is therefore fast and viable.
Wilson
L
L
Cedars-Sinai Medical Center, Division of Reproductive Medicine, Los Angeles, CA 90048.
Quinn
P
P
D000818:Animals; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000187:drug effects / Q000502:physiology*; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005615:Freezing; D051379:Mice; D008810:Mice, Inbred C57BL; D011309:Preservation, Biological / Q000379:methods*; D011409:Propylene Glycols / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors
D003451:Cryoprotective Agents; D011409:Propylene Glycols; D013395:Sucrose; D004121:Dimethyl Sulfoxide