Patent ID: 8293462

Claim:
A method of preserving mammalian early embryos or embryonic stem cells by vitrification, comprising: pretreating mammalian early embryos or embryonic stem cells in a pretreatment solution comprising a first phosphate buffer comprising a polyhydric alcohol constituent wherein said polyhydric alcohol constituent of the first phosphate buffer consists of 10% to 15% (v/v) propylene glycol; adding a solution for preservation by vitrification, said solution comprising a second phosphate buffer comprising polyhydric alcohol constituents, wherein said polyhydric alcohol constituents of said second phosphate buffer consist of (1) 10% to 15% (v/v) propylene glycol and (2) 25% to 35% (v/v) ethylene glycol, and wherein said second phosphate buffer further comprises 15% to 25% (v/v) Percoll® and 0.2 M to 0.5 M sucrose, to the mammalian early embryos or embryonic stem cells in the pretreatment solution to obtain a mixture wherein the volume ratio of the pretreatment solution to the solution for preservation by vitrification is 1:99 to 20:80; and cryopreserving the mammalian early embryos or embryonic stem cells in the mixture of the pretreatment solution and the solution for preservation by vitrification.