blastocysts, remove them from the tip, place them in the droplet of TS (drop A), and connect immediately with the first
droplet of DS (drop B) for 3 min.
10. Then connect with the second droplet of DS (drop C) for
another 2 min.
11. Then transfer the blastocysts to a fresh drop of 0.5 M sucrose
(drop D), and connect immediately with the first drop of WS
(drop E) for 3 min. Then connect with the second drop of WS
(drop F) for another 2 min (see Note 11).
12. Finally dilute and rinse through a series of two wash drops of
WS (G to H).
13. Place the blastocysts into a transfer culture dish, and put it in
the incubator for subsequent culture.
14. Record the survival and appearance of all blastocysts. Update
log with warm data, and notify the physician of result (see Note
12).
4 Notes
1. Special care must be given to the selection of the carriers. It is
necessary to use the types of carrier or vessel material with rapid
heat transfer that also support the process of uniform heat
exchange to achieve higher cooling rates.
2. In addition, although no report of contamination in human
IVF exists, the user should be encouraged to choose a closed
carrier system, which will work for blastocysts without any
problems.
3. To minimize the toxicity of the cryoprotectant, a stepwise
exposure of cells to pre-cooled concentrated solutions (around
room temperature of 24
C) is recommended.
4. Utilizing higher concentrations of cryoprotectant allows
shorter exposure times to the cryoprotectant—but be careful—the toxicity of the cryoprotectants might arise with their
concentration. Because almost all cryoprotectants are toxic to
some extent, it is important to carefully monitor the duration
of exposure to the final cryoprotectant before plunging into
liquid nitrogen.
5. To facilitate vitrification by even higher cooling rates, it is also
necessary to minimize the volume of the vitrification solution
(VS) as much as practical (preferable less than 1 μL). From this
point of view, it is very important to use a small pulled pipette.
Furthermore, by collecting the cells on one place and loading
not more as four cells at the same time in the pipette, it is
possible to keep the volume small. However, if the load of
Vitrification of Human Blastocysts
513
droplet of DS (drop B) for 3 min.
10. Then connect with the second droplet of DS (drop C) for
another 2 min.
11. Then transfer the blastocysts to a fresh drop of 0.5 M sucrose
(drop D), and connect immediately with the first drop of WS
(drop E) for 3 min. Then connect with the second drop of WS
(drop F) for another 2 min (see Note 11).
12. Finally dilute and rinse through a series of two wash drops of
WS (G to H).
13. Place the blastocysts into a transfer culture dish, and put it in
the incubator for subsequent culture.
14. Record the survival and appearance of all blastocysts. Update
log with warm data, and notify the physician of result (see Note
12).
4 Notes
1. Special care must be given to the selection of the carriers. It is
necessary to use the types of carrier or vessel material with rapid
heat transfer that also support the process of uniform heat
exchange to achieve higher cooling rates.
2. In addition, although no report of contamination in human
IVF exists, the user should be encouraged to choose a closed
carrier system, which will work for blastocysts without any
problems.
3. To minimize the toxicity of the cryoprotectant, a stepwise
exposure of cells to pre-cooled concentrated solutions (around
room temperature of 24
C) is recommended.
4. Utilizing higher concentrations of cryoprotectant allows
shorter exposure times to the cryoprotectant—but be careful—the toxicity of the cryoprotectants might arise with their
concentration. Because almost all cryoprotectants are toxic to
some extent, it is important to carefully monitor the duration
of exposure to the final cryoprotectant before plunging into
liquid nitrogen.
5. To facilitate vitrification by even higher cooling rates, it is also
necessary to minimize the volume of the vitrification solution
(VS) as much as practical (preferable less than 1 μL). From this
point of view, it is very important to use a small pulled pipette.
Furthermore, by collecting the cells on one place and loading
not more as four cells at the same time in the pipette, it is
possible to keep the volume small. However, if the load of
Vitrification of Human Blastocysts
513