2.2 Vitrification
1. Plastic straws (IMV, L’Aigle, France) (see Note 7).
2. Plugs for straws (IMV, L’Aigle, France) or sealing powder or
heat sealer (see Note 2).
3. Pulled glass pipettes, denudation glass or plastic pipettes.
4. Tissue culture dishes (Falcon, Becton and Dickinson Co.,
USA).
5. Dissecting microscope.
6. Thermocouple.
7. Device to hold straw horizontally above liquid nitrogen vapor
without covering area of straw containing oocytes (see Note 8).
8. Hotplate set at 37
C.
9. Liquid nitrogen dewars, preferably at least two storage dewars
(see Note 3), plus a container capable of holding liquid nitrogen and accommodating a straw held horizontally.
10. Scalpel.
11. 3Â 1 mL syringe and 2Â needle.
12. Heated water bath set at 20
C.
13. Safety equipment, e.g., cryo gloves, face shield, and oxygen
depletion monitor.
14. Heated gassed incubator.
15. Oocyte culture medium, e.g., for human oocytes fertilization
medium (Cook IVF, Brisbane, Australia) and for murine
oocytes Tyrode’s medium (Invitrogen, Paisley, UK).
16. Dulbecco’s phosphate-buffered solution (PBS; Invitrogen,
Paisley, UK) for human oocytes supplemented with 30% protein supplement, e.g., plasma protein supplement (Baxter AG,
Vienna, Austria) or serum protein supplement (Pacific Andrology, CGA/Diasint, Florence, Italy) for mouse oocytes supplemented with 5% heat-inactivated fetal bovine serum
(Invitrogen, Paisley, UK).
17. VS solutions: 6 M dimethyl sulfoxide (Me 2 SO) plus 1 mg/mL
polyethylene glycol (PEG; MW 8000) added to 4Â strength
PBS medium without CaCl 2 (see Note 9). The solution (for a
volume of 10 mL) should be made up by adding 1 mL of
distilled water to 2.5 mL of 4Â PBS prior to adding the
Me 2 SO (4.69 mL) and PEG (1 mL of 10 mg/mL aqueous
stock). Then add protein supplement (e.g., 0.5 mL FBS) and
make up almost to volume with water prior to adding CaCl 2
(100 μL of aqueous stock solution of 15.9 g/L). Finally, add
distilled water to make up to the final volume (10 mL in this
case). Keep at room temperature until required. This solution
will be referred to as VSDP (see Note 10). Make up dilutions of
25% and 65% VSDP using PBS containing protein supplement
as required.
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Victoria Keros and Barry J. Fuller
1. Plastic straws (IMV, L’Aigle, France) (see Note 7).
2. Plugs for straws (IMV, L’Aigle, France) or sealing powder or
heat sealer (see Note 2).
3. Pulled glass pipettes, denudation glass or plastic pipettes.
4. Tissue culture dishes (Falcon, Becton and Dickinson Co.,
USA).
5. Dissecting microscope.
6. Thermocouple.
7. Device to hold straw horizontally above liquid nitrogen vapor
without covering area of straw containing oocytes (see Note 8).
8. Hotplate set at 37
C.
9. Liquid nitrogen dewars, preferably at least two storage dewars
(see Note 3), plus a container capable of holding liquid nitrogen and accommodating a straw held horizontally.
10. Scalpel.
11. 3Â 1 mL syringe and 2Â needle.
12. Heated water bath set at 20
C.
13. Safety equipment, e.g., cryo gloves, face shield, and oxygen
depletion monitor.
14. Heated gassed incubator.
15. Oocyte culture medium, e.g., for human oocytes fertilization
medium (Cook IVF, Brisbane, Australia) and for murine
oocytes Tyrode’s medium (Invitrogen, Paisley, UK).
16. Dulbecco’s phosphate-buffered solution (PBS; Invitrogen,
Paisley, UK) for human oocytes supplemented with 30% protein supplement, e.g., plasma protein supplement (Baxter AG,
Vienna, Austria) or serum protein supplement (Pacific Andrology, CGA/Diasint, Florence, Italy) for mouse oocytes supplemented with 5% heat-inactivated fetal bovine serum
(Invitrogen, Paisley, UK).
17. VS solutions: 6 M dimethyl sulfoxide (Me 2 SO) plus 1 mg/mL
polyethylene glycol (PEG; MW 8000) added to 4Â strength
PBS medium without CaCl 2 (see Note 9). The solution (for a
volume of 10 mL) should be made up by adding 1 mL of
distilled water to 2.5 mL of 4Â PBS prior to adding the
Me 2 SO (4.69 mL) and PEG (1 mL of 10 mg/mL aqueous
stock). Then add protein supplement (e.g., 0.5 mL FBS) and
make up almost to volume with water prior to adding CaCl 2
(100 μL of aqueous stock solution of 15.9 g/L). Finally, add
distilled water to make up to the final volume (10 mL in this
case). Keep at room temperature until required. This solution
will be referred to as VSDP (see Note 10). Make up dilutions of
25% and 65% VSDP using PBS containing protein supplement
as required.
444
Victoria Keros and Barry J. Fuller