title: Cooling of embryonic cells, isolated blastoderms, and intact embryos of the zebra fish Brachydanio rerio to -196 degrees C.
abstract: Single cells from the developing embryo of the zebra fish survive freezing when protected with 1 M DMSO and cooled to -196 degrees C in two steps. Cell survival drops from 85 to 26% when clumps of 5-10 cells are similarly frozen, and to 2% when isolated blastoderms are treated in the same way. This drastic decrease in survival is interpreted as an example of the "scale-up problem," in which diffusional barriers prevent cryoprotectant equilibration and osmotic dehydration in large cell assemblanges. Isolated blastoderms develop considerably in culture, and retain some of this ability following cooling to -25 degrees C after protection with DMSO or glycerol. Intact embryos protected with high concentrations of glycerol (2.8 M) tolerate slow cooling to -196 degrees C surprisingly well, with most of the embryonic cells morphologically intact and actively extruding lobopodia. Glycerol could, however, only be removed from cells by disrupting the embryo so that diffusional barriers were removed. DMSO (2.8 M) was ineffective in preserving embryos or cells cooled to -196 degrees C.
mesh_terms: D000818:Animals; D003451:Cryoprotective Agents / Q000008:administration & dosage*; D004121:Dimethyl Sulfoxide / Q000008:administration & dosage; D004625:Embryo, Nonmammalian / Q000502:physiology*; D005399:Fishes / Q000196:embryology*; D005615:Freezing; D005990:Glycerol / Q000008:administration & dosage; D011309:Preservation, Biological; D013997:Time Factors
chemical_list: D003451:Cryoprotective Agents; D005990:Glycerol; D004121:Dimethyl Sulfoxide