2.2 Tools for Oocyte/
Zygote Vitrification
and Storage
1. A stereo microscope to visualize oocytes/zygotes during
handling (recommended: Olympus SZX16, Tokyo, Japan).
2. Fine glass pipettes with an inner diameter of 230–280 μm or
150–190 μm with a mouth piece to handle cumulus-enclosed
oocytes (CEOs) or zygotes, respectively. Such pipettes can be
forged from sterilized Pasteur pipettes over a gas or alcohol
burner. Three pipettes are required: (a) to move oocytes/
zygotes from collection (or culture) dishes to BM, (b) to
move oocytes/zygotes from base medium to EM, and (c) to
move oocytes/zygotes from EM to VM and to make microdrops (henceforth called “dropping” pipette). The ideal length
and shape of pipettes might depend on the technician. Straightpulled pipettes seem more feasible than bent ones to drop VM
on the cooling surface (see Note 7).
3. A 4-well culture dish (Nunc MultiDishes, Thermo Fisher Scientific, Waltham, MA, USA) for the vitrification of each group
of up to 100 oocytes/zygotes to be preserved together.
4. A 2 mL cryogenic vials (cryo-vial) for each oocyte/zygote lot
(e.g., Corning) for each group of up to 100 oocytes/zygotes to
be preserved together.
5. Cryoware marker (cryo-pen) (e.g., Nalge Nunc Int.).
6. Tube holder canes. Before using, make sure the cryo-vials can
be fit in them easily.
7. Two long (25–30 cm) forcipes for the handling of cryo-vials.
8. A fine tweezer (10–11 cm) to handle vitrified microdrops. For
firm grip, consider the use of a cross-action tweezer (such as
Dumont N7).
9. A Styrofoam box (approximate inner parameters:
widthÂlengthÂdepth of 12 Â 20 Â 13 cm).
10. Liquid nitrogen (LN).
11. A cooling surface prepared from aluminum foil. Parameters
and folding are shown in Fig. 1. Two types of devices can be
prepared; for solid surface vitrification a single cooling surface
(see Fig. 1a), for the traditional microdrop vitrification a
divided cooling surface (see Fig. 1b). A divided cooling surface
is useful to prevent the accidental fusion of microdrops. Before
folding, wipe the aluminum foil with 99% ethanol. Handle the
aluminum foil with sterile gloved hands.
12. Timer.
13. Sixty or 100 mm Petri dishes.
14. Injection needles or drawing pins (3 pieces, any type) to fix the
cooling surface.
Porcine Oocyte Vitrification
457
Zygote Vitrification
and Storage
1. A stereo microscope to visualize oocytes/zygotes during
handling (recommended: Olympus SZX16, Tokyo, Japan).
2. Fine glass pipettes with an inner diameter of 230–280 μm or
150–190 μm with a mouth piece to handle cumulus-enclosed
oocytes (CEOs) or zygotes, respectively. Such pipettes can be
forged from sterilized Pasteur pipettes over a gas or alcohol
burner. Three pipettes are required: (a) to move oocytes/
zygotes from collection (or culture) dishes to BM, (b) to
move oocytes/zygotes from base medium to EM, and (c) to
move oocytes/zygotes from EM to VM and to make microdrops (henceforth called “dropping” pipette). The ideal length
and shape of pipettes might depend on the technician. Straightpulled pipettes seem more feasible than bent ones to drop VM
on the cooling surface (see Note 7).
3. A 4-well culture dish (Nunc MultiDishes, Thermo Fisher Scientific, Waltham, MA, USA) for the vitrification of each group
of up to 100 oocytes/zygotes to be preserved together.
4. A 2 mL cryogenic vials (cryo-vial) for each oocyte/zygote lot
(e.g., Corning) for each group of up to 100 oocytes/zygotes to
be preserved together.
5. Cryoware marker (cryo-pen) (e.g., Nalge Nunc Int.).
6. Tube holder canes. Before using, make sure the cryo-vials can
be fit in them easily.
7. Two long (25–30 cm) forcipes for the handling of cryo-vials.
8. A fine tweezer (10–11 cm) to handle vitrified microdrops. For
firm grip, consider the use of a cross-action tweezer (such as
Dumont N7).
9. A Styrofoam box (approximate inner parameters:
widthÂlengthÂdepth of 12 Â 20 Â 13 cm).
10. Liquid nitrogen (LN).
11. A cooling surface prepared from aluminum foil. Parameters
and folding are shown in Fig. 1. Two types of devices can be
prepared; for solid surface vitrification a single cooling surface
(see Fig. 1a), for the traditional microdrop vitrification a
divided cooling surface (see Fig. 1b). A divided cooling surface
is useful to prevent the accidental fusion of microdrops. Before
folding, wipe the aluminum foil with 99% ethanol. Handle the
aluminum foil with sterile gloved hands.
12. Timer.
13. Sixty or 100 mm Petri dishes.
14. Injection needles or drawing pins (3 pieces, any type) to fix the
cooling surface.
Porcine Oocyte Vitrification
457