9. Remove embryos from culture dishes using a stripper tip into
the HTF-HEPES (drop 1), gently aspirating to remove any
traces of culture media.
10. Pipette from mHTF or WS (drop 1) to the other drop of
mHTF or WS (drop 2), and immediately merge it with the
first drop of ES (drop 3). Set the timer for 5 min.
11. When the time is up, transfer the embryos to the remaining
drop of ES (drop 4). Set the timer for 3 min. Place the embryos
on the top of the drop, and let them settle to the bottom.
12. Then load the blastocysts in a VS back-loaded stripper tip, and
rinse through the four droplets of VS (drops 5–8), between
each droplet clean your tip.
13. Placement into the VS and loading of the Cryotop should take
less than 1 min, so that the total incubation time in 15% VS is
30 s. After 30 s, gently transfer them to the tip of the HSV by
using a stripper tip to load the blastocysts in as small volume
(less than 0.5 μL) as possible onto the edge of the stick (see
Note 5).
14. Visually confirm placement (see Note 6).
15. Before loading, apply the label to the open end of the empty
straw. Load the HSV stick into the empty straw, the side with
the embryos first. Use the blue handle to make sure the stick is
in as far as it goes. Then, using the heat sealer, seal the open end
of the stick and plunge the whole straw into the LN 2 . Place the
straw in a pre-cooled aluminum cane for further storage (see
Notes 7 and 8).
16. Store at the cane in a nitrogen tank.
17. Make sure to record cane location on the freezing worksheet
and cryo inventory log.
18. Complete all paperwork, and recheck that all vial locations are
logged into the embryo inventory.
3.2 Stepwise
Blastocyst Warming
Procedure
Important to be mentioned is that regardless of the day of cryopreservation of the embryo (whether day 5, 6, or 7), at thawing,
blastocysts should be treated as if they had been frozen on the fifth
day of development. To remove the cryoprotectants, blastocysts
need to be warmed and diluted in a three-step process. With the
HSV submerged in LN 2 , the inner straw should be removed, and
then the carrier with the blastocysts can be removed from the LN 2
and placed directly into a pre-warmed (37
C) 200 μL drop of
1.0 M sucrose on a 90 mm dish lid surface (see Note 9). Blastocysts
can be picked up directly from the HSV, placed in a fresh 50 μL
drop of 1.0 M sucrose at 24
C, and immediately connected with a
50 μL drop of 0.5 M sucrose. After 3 min connect with another
50 μL drop of 0.5 M sucrose for 2 min. Then blastocysts can be
Vitrification of Human Blastocysts
511
the HTF-HEPES (drop 1), gently aspirating to remove any
traces of culture media.
10. Pipette from mHTF or WS (drop 1) to the other drop of
mHTF or WS (drop 2), and immediately merge it with the
first drop of ES (drop 3). Set the timer for 5 min.
11. When the time is up, transfer the embryos to the remaining
drop of ES (drop 4). Set the timer for 3 min. Place the embryos
on the top of the drop, and let them settle to the bottom.
12. Then load the blastocysts in a VS back-loaded stripper tip, and
rinse through the four droplets of VS (drops 5–8), between
each droplet clean your tip.
13. Placement into the VS and loading of the Cryotop should take
less than 1 min, so that the total incubation time in 15% VS is
30 s. After 30 s, gently transfer them to the tip of the HSV by
using a stripper tip to load the blastocysts in as small volume
(less than 0.5 μL) as possible onto the edge of the stick (see
Note 5).
14. Visually confirm placement (see Note 6).
15. Before loading, apply the label to the open end of the empty
straw. Load the HSV stick into the empty straw, the side with
the embryos first. Use the blue handle to make sure the stick is
in as far as it goes. Then, using the heat sealer, seal the open end
of the stick and plunge the whole straw into the LN 2 . Place the
straw in a pre-cooled aluminum cane for further storage (see
Notes 7 and 8).
16. Store at the cane in a nitrogen tank.
17. Make sure to record cane location on the freezing worksheet
and cryo inventory log.
18. Complete all paperwork, and recheck that all vial locations are
logged into the embryo inventory.
3.2 Stepwise
Blastocyst Warming
Procedure
Important to be mentioned is that regardless of the day of cryopreservation of the embryo (whether day 5, 6, or 7), at thawing,
blastocysts should be treated as if they had been frozen on the fifth
day of development. To remove the cryoprotectants, blastocysts
need to be warmed and diluted in a three-step process. With the
HSV submerged in LN 2 , the inner straw should be removed, and
then the carrier with the blastocysts can be removed from the LN 2
and placed directly into a pre-warmed (37
C) 200 μL drop of
1.0 M sucrose on a 90 mm dish lid surface (see Note 9). Blastocysts
can be picked up directly from the HSV, placed in a fresh 50 μL
drop of 1.0 M sucrose at 24
C, and immediately connected with a
50 μL drop of 0.5 M sucrose. After 3 min connect with another
50 μL drop of 0.5 M sucrose for 2 min. Then blastocysts can be
Vitrification of Human Blastocysts
511