Therefore, we composed new vitrification solutions (EFSc solutions) in which the concentration of sucrose was increased to 3.1 mol/kg water. The increase in the sucrose concentration increases not only the molality (moles/kilogram water) of sucrose but also that of ethylene glycol, because the increase in the volume of sucrose decreases the volume of water. Eventually, the osmolality of EFSc solution was increased greatly (Table 1). When two-cell embryos (ICR mice) were vitrified with EFS30c and EFS40c, a high proportion (86%-88%) survived as expected after being kept at —80°C for 4 days before recovery (Table 2). In a preliminary experiment, we composed EFSb solutions with the FSb solution, which contained 1.0 M sucrose. However, EFS30b and EFS40b were much less effective than EFS30c and EFS40c (data not shown). With EFS35c and EFS40c, a high proportion (82%-83%) of embryos survived after being kept at —80°C even for 10 days. With EFS30c, on the other hand, the swelling rate increased (26%) and the survival rate decreased when embryos were kept at —80°C for 7 days (Table 3), probably because the osmolality of the solution was not sufficiently high to prevent the formation of intracellular ice. In EFS30c, EFS35c, and EFS40c, the molality of sucrose is the same (3.1 mol/kg water), but the molality of ethylene glycol is different (15.8, 19.9, and 24.6 mol/kg water, respectively; Table 1).

Considering the practical applications of the new method, we used two-cell embryos, because mouse embryos are most commonly cryopreserved at this stage, especially for the preservation of genetically engineered variants. We tried to find suitable conditions for embryos of ICR mice (Tables 2-4) and then to confirm the efficacy with embryos of C57BL/6J mice (Table 5), because this strain is widely used for genetic engineering.

When two-cell embryos of C57BL/6J mice were vitrified with EFS35c or EFS40c and then kept at —80°C for 4 days before recovery, the survival rate remained as high (88%-89%; Table 5) as that of fresh control embryos (91%; Table 5). In addition, we have confirmed that a high proportion of two-cell embryos of C57BL/6J mice vitrified and kept at —80°C for 4 days survive after being recooled in LN2 (87%-88%; Table 5). When the embryos were transferred to recipient mice, the rates of implantation and offspring (95% and 75%, respectively) were as high as those of fresh control (93% and 68%, respectively). This demonstrates that an equilibrium vitrification method was developed in the present study.

In the mouse, a large number of transgenic and knockout variants have been produced, many of which are preserved as embryos in LN2. Consequently, the need to transport cryopreserved embryos is increasing. To maintain their viability during transportation, embryos should be kept at below the glass transition temperature of the cytoplasm (approximately —130°C), preferably in LN2 (—196°C). In most cases, however, sending specimens in LN2 is not allowed, and thus cryopreserved embryos are transported in a dry shipper, a large Dewar flask designed for the shipment of specimens in LN2 in the gas phase. Unfortunately, the dry shipper is large, heavy, and expensive. Therefore, an advantage of equilibrium vitrification would be that cryopreserved embryos can be transported using dry ice and can be represerved in LN2.

In conclusion, we have developed a novel method by which embryos are vitrified with minimal supercooling (i.e., in near equilibrium). This was a missing approach (Fig. 1), and it would become a supreme method because it has clear advantages over existing methods. Practically, the method is as simple and quick as current vitrification. In addition, it has advantages that current vitrification does not have. Embryos preserved by this method are less susceptible to damage during handling. This method would enable the temporary evacuation of vitrified samples into a freezer at —80°C in an emergency or for the arrangement of cryopreserved samples, as well as the handy short-range transportation of vitrified embryos using dry ice.

Category: Equilibrium Vitrification / Tags: liquid nitrogen, mammalian embryos, promotes dehydration