An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.
Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4°C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.
The overall scheme of the experiment is shown in Fig. 1.
1. Reagent Preparation
M.W. | mM | mg/100ml | |
NaCl | 58.4 | 136.98 | 800.0 |
KCl | 74.6 | 2.68 | 20.0 |
KH2PO4 | 136.1 | 1.47 | 20.0 |
Na2HPO4.12H2O | 358.14 | 8.04 | 288.1 |
MgCl2,6H2O | 203.3 | 0.49 | 10.0 |
Glucose | 180.2 | 5.56 | 100.0 |
Na pyruvate | 110 | 0.33 | 3.6 |
CaCl2,2H2O | 147 | 0.9 | 13.2 |
Penicillin G | 6.0 (approx) |
Ficoll 70 | 6.0 g |
Sucrose | 3.424 g |
BSA | 42.0 mg |
EFS20 solution | EFS40 solution | |
Ethylene glycol | 1 ml | 2 ml |
FS solution | 4 ml | 3 ml |
2. Vitrification of 2-cell Mouse Embryos
3. Thawing Vitrified 2-cell Mouse Embryos
4. Representative Results:
In vitro– and in vivo-development of embryos after thawing is presented in Tables 1 and 2. The advantages of this protocol are the high survivability of embryos after thawing and its broad applicability to different strains of mice.
Strain | Total No. of tubes | No. of embryos vitrified | Recovered (No. (%)) | Morphologically normal (No. (%)) | Development to blastocysts (No. (%)) |
C57BL/6J | 20 | 400 | 397 (99) | 394 (99) | 342 (87) |
BALB/cA | 15 | 300 | 296 (99) | 282 (95) | 238 (84) |
ICR | 24 | 480 | 474 (99) | 443 (93) | 398 (90) |
Table 1. In vitro-development of vitrified-thawed embryos in common mouse strains
Condition of embryos | No. of recipient females | No. of embryos transferred | Implantation sites (No. (%)) | Live offspring (No. (%)) |
Fresh | 12 | 180 | 141 (78.3) | 110 (61.1) |
Vitrified | 16 | 242 | 202 (83.5) | 125 (51.7) |
Table 2. In vivo-development of vitrified-thawed embryos in C57BL/6J mice.
Figure 1. The overall scheme of the experiment including equilibration, vitrification, and thawing of embryos.
Figure 2. The morphology of embryos at each step of thawing.
Figure 3. Thawing procedure of embryos. All procedures are performed at room temperature. (A), Add 850 μl of TS1 (37°C) into a cryotube and transfer the entire volume of the solution in the cryotube onto a plastic dish. At this time, the embryos look swollen as shown in Fig. 2B. (B), Spread the solution over the surface of the dish by gentle shaking. (C), Place three 50 μl drops of TS2 on the plastic dish. (D), Transfer the embryos to the first drop of TS2. After 3 min, the embryos look shurunken as shown in Fig. 2C. (E), Transfer them serially into the remaining TS2 drops and then to the culture medium.
Since the first report of mouse embryo vitrification by Rall and Fahy in 19852, several technical improvements have been made to increase the survivability of embryos after thawing. One of the most successful modifications was achieved by use of ethylene glycol as a cryoprotectant because of its low toxicity and high membrane-permeability. Such advantages enable us to handle freezing embryos at room temperature4; other vitrification methods require embryo handing at cooler temperatures and are not always applicable to some strains of mice including BALB/c6. The first ethylene glycol-based vitrification was developed by Kasai et al. in 19905,7. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. Therefore, the vitrification method described here may be applicable to many laboratories using mice as research models. The same method can also be used for mouse embryos at the morula and blastocyst stages8 and rat embryos9. However, we have recently found that the quality of cryotubes may affect the survival rates of embryos after freezing-thawing. Thus, it is essential to examine the inside surface of the cryotubes for its smoothness before vitrifying embryos (e.g.; see at Table of specific reagents and equipment).
Vitrification methods have many advantages over conventional slow freezing methods, but they inherently have a disadvantage in respect to transportation purpose. As vitrified embryos should be kept at below -120°C to maintain their viability, dry shippers are generally used for their safe transportation. Dry shippers are heavy and bulky, and their round-trip is expensive, especially for international transportation. We are now currently developing a new vitrification method by which vitrified embryos can be stored at dry-ice temperature (about -80°C) for at least 7 days10. This method should be the vitrification of the next generation.
The authors have nothing to disclose.
This study was conducted in cooperation with the National BioResource Project, the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Name of the reagent | Company | Catalogue number | Comments (optional) |
Bovine serum albumin | Merck Biosciences (Calbiochem) | 12657 | |
Ethylene glycol | Wako Pure Chemical Industries | 058-00986 | |
Ficoll 70 | GE Healthcare | 17-0310-10 | |
Glucose | Wako Pure Chemical Industries | 041-00595 | |
NaCl | Wako Pure Chemical Industries | 191-01665 | |
KCl | Wako Pure Chemical Industries | 163-03545 | |
KH2PO4 | Wako Pure Chemical Industries | 169-04245 | |
Na2HPO4·12H2O | Wako Pure Chemical Industries | 500-04195 | |
MgCl2 ·6H2O | Wako Pure Chemical Industries | 135-00165 | |
CaCl2 ·2H2O | Wako Pure Chemical Industries | 031-00435 | |
Penicillin G | Sigma-Aldrich | P-4687 | |
Sodium pyruvate | Sigma-Aldrich | P-8574 | |
Sucrose | Wako Pure Chemical Industries | 196-00015 | |
M16 medium | Sigma-Aldrich | M7292 | |
M2 medium | Sigma-Aldrich | M7167 | |
Cryotube | Sumitomo Bakelite | MS-4501 | “Cryogenic Vial” |