"Two-step" freezing of individual blastomeres from mouse eight-cell embryos

Eight-cell embryos recovered from superovulated C57BL/6J mice were disaggregated into individual 1/8 blastomeres. For cryopreservation of blastomeres a simple "two-step" technique based on direct transfer of blastomeres into the final concentration of DMSO (0.5 to 1.5 M) and exposure of plastic straws with blastomeres to -25 degrees C for 10 min before immersion into liquid nitrogen was used. The samples stored for 2 to 60 days were thawed at different temperature (20 to 80 degrees C) and intact 1/8 blastomeres were transferred into DMSO-free medium. The survival of blastomeres in individual groups varied from 52% to 90% according to the combination of DMSO concentration, exposure time to DMSO prior to freezing and temperature of thawing bath. Intact frozen-thawed 1/8 blastomeres were able to undergo cleavage and aggregates constructed from 4, 6, 7, and 8 blastomeres developed throughout preimplantation period. Of the aggregates constructed 1 to 2 h after thawing, 78% to 93% reached the blastocyst stage within 54 h of in vitro culture. Possible use of frozen-thawed 1/8 blastomeres in nuclear transplantation experiments and genetic manipulation on mammalian embryos is discussed.