A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos.
The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3 mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures. A new vitrification method using P10 and PEPeS was tested using rat embryos. The survival rate of vitrified embryos after exposure to P10 for 120, 300, or 600 s ranged from 95.9% to 98.3%. The fetal developmental rate ranged from 57.7% to 65.2%, which was not significantly different from that of fresh embryos. The experimental results indicated that vitrification using a combination of P10 and PEPeS was suitable for cryopreservation of rat early stage embryos.
Central Institute for Experimental Animals, Kawasaki 210-0821, Japan. Electronic address: etoh@ciea.or.jp.
Tomoo
T
Eto
In-Vivo Science, Inc., Kawasaki 210-0821, Japan.
Riichi
R
Takahashi
Central Institute for Experimental Animals, Kawasaki 210-0821, Japan.
Tsutomu
T
Kamisako
Central Institute for Experimental Animals, Kawasaki 210-0821, Japan.
Kyoji
K
Hioki
Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan.
Yusuke
Y
Sotomaru
D000818:Animals; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D047108:Embryonic Development / Q000187:drug effects*; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D005990:Glycerol / Q000494:pharmacology; D008297:Male; D009994:Osmolar Concentration; D011205:Povidone / Q000494:pharmacology; D019946:Propylene Glycol / Q000494:pharmacology; D051381:Rats; D012822:Silicon Dioxide / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology; D058989:Vitrification
D003451:Cryoprotective Agents; D013395:Sucrose; C016039:Percoll; D019946:Propylene Glycol; D012822:Silicon Dioxide; D019855:Ethylene Glycol; D011205:Povidone; D005990:Glycerol; D004121:Dimethyl Sulfoxide
Cryopreservation; Cryoprotectant solution; P10; PEPeS; Rat embryos; Strain preservation; Vitrification