FIG. 1. Schematic representation of an embryo (circle) during nonequilibrium slow freezing, equilibrium slow freezing, nonequilibrium vitrification, and equilibrium vitrification. Darker representation shows higher osmolality. Small hexagons show ice crystals. Black lines show rapid cooling, and dotted lines show slow cooling. CPA, cryoprotectant.
For the cryopreservation of mammalian embryos, various methods have been developed. The method most widely used is interrupted slow freezing, where embryos are cooled slowly to around —30°C using a programmable freezer before being cooled rapidly with liquid nitrogen (LN2). The embryos are preserved with considerable supercooling (in nonequilibrium) because slow cooling that promotes dehydration is interrupted, and thus the osmolality of the cytoplasm is not sufficiently high (Fig. 1). Above the glass transition temperature of the cytoplasm (approximately —130°C), the vitrified (solid) cytoplasm would turn to liquid, and the supercooled cytoplasm could devitrify. Devitrification of the cytoplasm is the formation of intracellular ice and is lethal to embryos. To prevent this, the sample needs to be warmed rapidly.
With original slow freezing, embryos are cooled slowly to approximately —70°C before being cooled in LN2. Embryos frozen by this method would be dehydrated sufficiently, and thus cryopreserved in LN2 with a minimal extent of supercooling (in near-equilibrium; Fig. 1). With this equilibrium freezing, embryos survive not only after slow warming but also after preservation at —80°C for as long as 6 mo (M. Yokoyama, personal communication). http://birthcontroltab.com/buy-alesse-online.html
Consequently, the original slow freezing has several advantages; 1) embryos are less likely to be damaged during handling for warming, 2) frozen embryos can be evacuated in a freezer at —80°C for a certain period in case of trouble with the LN2 tank or for arrangement of samples, and 3) frozen embryos can be transported short distances using dry ice (—79°C). However, slow freezing has disadvantages in that it requires ice seeding and a programmable freezer for slow cooling, and it takes a long time.
Since the pioneering reports on the vitrification of mouse embryos, the method has been modified and become an alternative to slow freezing for the cryopreservation of mammalian embryos, because vitrification has many advantages; it requires neither ice seeding nor slow cooling nor a programmable freezer, and thus cooling is instant. In addition, vitrification is expected to achieve a high rate of survival because of the absence of ice. With current vitrification, however, embryos are vitrified with considerable supercooling, and thus cryopreserved in nonequilibrium as in interrupted slow freezing (Fig. 1).