4. Calibrate the dropping pipette by suckling up 2 μL of vitrification solution and marking the edge of the medium at the
outside of the tip with a cryo-pen (see Fig. 2).
5. Prepare one small forceps for handling microdrops and two
large forcipes for handling cryo-vials.
6. Prepare cryo-vials and canes. One cryo-vial is used for each
oocyte lot. Label them with cryo-pen before use.
7. Prepare an equilibration dish for each oocyte lot of up to
100 oocytes using a 4-well culture dish as follows: add
500 μL of BM in the first well; 500 μL aliquots of EM in the
second and third wells; and approximately 1 mL of the VM in
the fourth well (see Fig. 3). Keep the dish at room temperature.
8. Prepare the Styrofoam box and LN.
3.2 Vitrification
and Storage
1. Place a group of oocytes/zygotes (up to 100, to be vitrified
together) into the first well of the vitrification dish in BM, and
wash them briefly (1–2 min).
2. Equilibration treatment: with another (second) transfer
pipette, transfer all oocytes/zygotes into the second well of
the equilibration dish in EM, and start the timer (0 min of
equilibration). After one brief washing in the second well,
transfer all oocytes/zygotes into the third well of the dish
(EM). The oocytes/zygotes will be kept in EM for 13–15 min.
Fig. 2 The optimum size, calibration, and loading of glass pipettes used for the placing of oocytes/zygotes in
droplets of VM on cooling surfaces. The optimum opening for immature cumulus-enclosed oocytes is
230–280 μm in diameter (D1). The optimum opening for denuded zygotes is 150–190 μm in diameter (D2).
To calibrate the pipette (a), first pick up 2 μL of VM without oocytes, and mark the edge with a cryo-pen, and
then empty the pipette. To load the pipette (b), oocytes/zygotes must be kept together tightly closed to each
other and moved as a group. Before placing the droplets, the tip of the pipette (approximately 10 mm from the
opening) should not contain oocytes, to avoid their sticking to the glass pipette during dropping
460
Tamas Somfai and Kazuhiro Kikuchi
outside of the tip with a cryo-pen (see Fig. 2).
5. Prepare one small forceps for handling microdrops and two
large forcipes for handling cryo-vials.
6. Prepare cryo-vials and canes. One cryo-vial is used for each
oocyte lot. Label them with cryo-pen before use.
7. Prepare an equilibration dish for each oocyte lot of up to
100 oocytes using a 4-well culture dish as follows: add
500 μL of BM in the first well; 500 μL aliquots of EM in the
second and third wells; and approximately 1 mL of the VM in
the fourth well (see Fig. 3). Keep the dish at room temperature.
8. Prepare the Styrofoam box and LN.
3.2 Vitrification
and Storage
1. Place a group of oocytes/zygotes (up to 100, to be vitrified
together) into the first well of the vitrification dish in BM, and
wash them briefly (1–2 min).
2. Equilibration treatment: with another (second) transfer
pipette, transfer all oocytes/zygotes into the second well of
the equilibration dish in EM, and start the timer (0 min of
equilibration). After one brief washing in the second well,
transfer all oocytes/zygotes into the third well of the dish
(EM). The oocytes/zygotes will be kept in EM for 13–15 min.
Fig. 2 The optimum size, calibration, and loading of glass pipettes used for the placing of oocytes/zygotes in
droplets of VM on cooling surfaces. The optimum opening for immature cumulus-enclosed oocytes is
230–280 μm in diameter (D1). The optimum opening for denuded zygotes is 150–190 μm in diameter (D2).
To calibrate the pipette (a), first pick up 2 μL of VM without oocytes, and mark the edge with a cryo-pen, and
then empty the pipette. To load the pipette (b), oocytes/zygotes must be kept together tightly closed to each
other and moved as a group. Before placing the droplets, the tip of the pipette (approximately 10 mm from the
opening) should not contain oocytes, to avoid their sticking to the glass pipette during dropping
460
Tamas Somfai and Kazuhiro Kikuchi