6. At 13 min of the equilibration treatment, pick up one group of
30–50 oocytes/zygotes from the third well (EM) with the
third (dropping) pipette, and wash them consecutively
3 times in one triplet of VM drops. From the last drop, pick
up all 30–50 oocytes with 2 μL of vitrification medium (use a
calibrated pipette), and instantaneously drop them on the surface of aluminum foil in 1–3 microdrops with repeated hand
flicks (see Notes 14 and 15). Then wash the vitrification pipette
in the second well of the equilibration dish to remove residual
cryoprotectant agents, pick up the second group of 30–50
oocytes/zygotes from the third well, and process them in the
same way. Treatment of oocytes with VM (i.e., from placing
them to the first drop of VM until making microdrops) should
be performed within 30–40 s. It is of the utmost importance
that treatment of oocytes/zygotes with the VM should not
reach 60 s [12]. Total treatment of oocytes/zygotes in EM
should not exceed 15 min.
7. Transparent (“glassy”) microdrops are considered successfully
vitrified (see Note 16) and are subsequently transferred into
cryo-vials. Open a cryo-vial, partially submerge it in LN in the
Styrofoam box with long forceps, and fill it with LN (see
Note 17) to at least half of its volume. Move vitrified droplets
into the cryo-vial with small forceps. Before touching the droplets always pre-cool the tip of the forceps in LN. Close the lid
of the cryo-vial with another long forceps, put it in a tubeholding cane, and store it submerged in LN in a LN tank.
3.3 Setting Up
for Warming
1. For each lot of vitrified oocytes to be warmed as a group,
preheat 2 mL of BM and 3 mL of WM to 38.0
C in a tube
heater (aluminum block).
2. Prepare a simple aluminum cooling surface and two glass transfer pipettes as described above.
3. Preheat a warming plate to 42
C for immature oocytes or to
38.0
C for zygotes (see Note 18) and the stage of the stereo
microscope to 38.0
C.
4. Prepare a warming dish by adding 2.5 mL of pre-warmed WM
into a 35 mm Petri dish. Keep it on the warm plate.
5. Prepare a re-hydration dish with stepwise-reduced sucrose concentration (see Fig. 4) using a 4-well culture dish as follows: first
add 500 μL of pre-warmed BM in each well of a 4-well dish.
Then add an additional 500 μL of pre-warmed WM (0.4 M
sucrose) to the first well and mix with pipette (1:1 dilution
ratio) to achieve a sucrose concentration of 0.2 M. Then pick
up 500 μL of medium from the first well, and add it to the
second well, and mix to achieve a sucrose concentration of
0.1 M. Finally, pick up 500 μL of medium from the second
462
Tamas Somfai and Kazuhiro Kikuchi
30–50 oocytes/zygotes from the third well (EM) with the
third (dropping) pipette, and wash them consecutively
3 times in one triplet of VM drops. From the last drop, pick
up all 30–50 oocytes with 2 μL of vitrification medium (use a
calibrated pipette), and instantaneously drop them on the surface of aluminum foil in 1–3 microdrops with repeated hand
flicks (see Notes 14 and 15). Then wash the vitrification pipette
in the second well of the equilibration dish to remove residual
cryoprotectant agents, pick up the second group of 30–50
oocytes/zygotes from the third well, and process them in the
same way. Treatment of oocytes with VM (i.e., from placing
them to the first drop of VM until making microdrops) should
be performed within 30–40 s. It is of the utmost importance
that treatment of oocytes/zygotes with the VM should not
reach 60 s [12]. Total treatment of oocytes/zygotes in EM
should not exceed 15 min.
7. Transparent (“glassy”) microdrops are considered successfully
vitrified (see Note 16) and are subsequently transferred into
cryo-vials. Open a cryo-vial, partially submerge it in LN in the
Styrofoam box with long forceps, and fill it with LN (see
Note 17) to at least half of its volume. Move vitrified droplets
into the cryo-vial with small forceps. Before touching the droplets always pre-cool the tip of the forceps in LN. Close the lid
of the cryo-vial with another long forceps, put it in a tubeholding cane, and store it submerged in LN in a LN tank.
3.3 Setting Up
for Warming
1. For each lot of vitrified oocytes to be warmed as a group,
preheat 2 mL of BM and 3 mL of WM to 38.0
C in a tube
heater (aluminum block).
2. Prepare a simple aluminum cooling surface and two glass transfer pipettes as described above.
3. Preheat a warming plate to 42
C for immature oocytes or to
38.0
C for zygotes (see Note 18) and the stage of the stereo
microscope to 38.0
C.
4. Prepare a warming dish by adding 2.5 mL of pre-warmed WM
into a 35 mm Petri dish. Keep it on the warm plate.
5. Prepare a re-hydration dish with stepwise-reduced sucrose concentration (see Fig. 4) using a 4-well culture dish as follows: first
add 500 μL of pre-warmed BM in each well of a 4-well dish.
Then add an additional 500 μL of pre-warmed WM (0.4 M
sucrose) to the first well and mix with pipette (1:1 dilution
ratio) to achieve a sucrose concentration of 0.2 M. Then pick
up 500 μL of medium from the first well, and add it to the
second well, and mix to achieve a sucrose concentration of
0.1 M. Finally, pick up 500 μL of medium from the second
462
Tamas Somfai and Kazuhiro Kikuchi