Cryopreservation of embryos
An enormous number of mouse strains have been created one after another by genetic manipulation and mutation in mice. The cryopreservation of embryos (fertilized eggs) from these strains will not only save space and money from maintaining the strains as live animals, but also protect them from microbial contamination and accidents, and preserve their genetic traits semi-permanently. Also, by transferring cryopreserved embryos to recipient females after thawing, it is possible to obtain the required number of animals systematically when needed. In addition, it can be transported in the state of frozen embryos and can be distributed with little possibility of microbial contamination.
As for the freezing method, each institution may introduce its own method, but it can be broadly divided into two types, the slow method, and the vitrification method. The slow method is a method in which embryos are immersed in a relatively low-concentration (low-toxicity) cryoprotectant and then performed over a period of one and a half hours using a program freezer. In the vitrification method, embryos are stored directly in liquid nitrogen using a denser concentration of antifreeze agents, but in a very shorter time (about 10 seconds to 10 minutes).
We used a vitrification method with both simplicity and high viability, in which embryos are introduced into EFS30 (30% ethylene glycol, 21% ficoll, 0.35Mol shoe cloth, 49% PB1) and then placed in liquid nitrogen in two minutes.
In experimental animals other than mice, the effectiveness of embryo freezing in rats and rabbits has been reported. In our laboratory, we have succeeded in obtaining pups from cryopreserved embryos from rodents (Ref. 1) and mastomys (Ref. 2), and we have also studied the Syrian hamster (Ref. 3). Successful cases in the Syrian hamster have been reported elsewhere recently. Sensitivity associated with the freezing operation varies depending on the animal species, including the mouse, its strain, and the stage of embryo development. Therefore, it is necessary to continue research on setting appropriate conditions for each animal species.
References
- Mochida K., Wakayama T., Nakayama K., Takano K., Noguchi Y., Yamamoto Y., Suzuki O., Ogura A. and Matsuda J. (1999) Successful cryopreservation of Mongolian gerbil embryos by vitrification. Theriogenology, 51, 171.
- Mochida K., Matsuda J., Noguchi Y., Yamamoto Y., Nakayama K., Takano K., Suzuki O. and Ogura A. (1998) Birth of pups by transfer of mastomys embryos cryopreserved by vitrification. Biology of Reproduction, 58, Supplement 1.
- Mochida K., Yamamoto Y., Noguchi Y., Takano K., Matsuda J. and Ogura A. (2000) Survival and subsequent in vitro development of hamster embryos after exposure to cryoprotectant solutions. Journal of Assisted Reproduction and Genetics, 17: 182-185.