title: Formation of extracellular and intracellular ice during warming of vitrified mouse morulae and its effect on embryo survival.
abstract: In vitrified solutions, ice can form during warming if the concentration of the cryoprotectant is insufficient. For the cryopreservation of cells, ice is innocuous when it remains outside the cell, but intracellular ice (ICI) is lethal. We tried to estimate the conditions in which ICI forms in vitrified mouse morulae during warming. The solutions for the experiments (EFS10-EFS50) contained 10-50% ethylene glycol plus Ficoll plus sucrose. When vitrified EFS20, EFS30, and EFS40 were kept at -80 degrees C, they remained transparent after 3 min, but turned opaque after 60 min (EFS20, EFS30) or 24h (EFS40). Morulae were vitrified with EFS solutions after exposure for 30-120 s at 25 degrees C. They were warmed by various methods and survival was assessed in culture. After rapid warming (control), survival was high with EFS30 (79-93%) and EFS40 (96-99%). After slow warming, survival decreased with both EFS30 (48-62%) and EFS40 (44-64%). This must be from the formation of ICI. To examine the temperature at which ICI formed during slow warming, vitrified embryos were kept at various sub-zero temperatures during warming. Survival with EFS30 and EFS40 decreased on keeping samples for 3 min at -80 (25-75%), -60 (7-49%), -40 (0-41%), or -20 degrees C (26-60%). When samples were kept at -80 degrees C for 24h, the survival decreased to 0-14%. These results suggest that ICI forms at a wide range of temperatures including -80 and -20 degrees C, more likely between -60 and -40 degrees C, and the ice forms not only quickly but also slowly.
authors.affiliation: Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan.
authors.forename: Bo
authors.initials: B
authors.lastname: Jin
authors.forename: Kenji
authors.initials: K
authors.lastname: Kusanagi
authors.forename: Makiko
authors.initials: M
authors.lastname: Ueda
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Seki
authors.forename: Delgado M
authors.initials: DM
authors.lastname: Valdez
authors.forename: Keisuke
authors.initials: K
authors.lastname: Edashige
authors.forename: Magosaburo
authors.initials: M
authors.lastname: Kasai
mesh_terms: D000818:Animals; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003460:Crystallization; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology; D000431:Ethanol / Q000737:chemistry; D019855:Ethylene Glycol / Q000494:pharmacology; D045604:Extracellular Fluid / Q000737:chemistry*; D005362:Ficoll / Q000494:pharmacology; D007053:Ice; D007424:Intracellular Fluid / Q000737:chemistry*; D051379:Mice; D008813:Mice, Inbred ICR; D009028:Morula / Q000166:cytology*; D009584:Nitrogen / Q000737:chemistry; D013395:Sucrose / Q000494:pharmacology; D044366:Transition Temperature
chemical_list: D003451:Cryoprotective Agents; D007053:Ice; D005362:Ficoll; D000431:Ethanol; D013395:Sucrose; D019855:Ethylene Glycol; D009584:Nitrogen