2 Materials
2.1 Slow Controlled
Rate Cooling
1. A controlled rate freezing machine (available from several
sources such as Planer plc, Middlesex, UK or Asymptote, Cambridge, UK) set to hold at 20
C, then cool at À2
C/min to
À7
C, allow for seeding (see Note 1), hold at À7
C for 10 min
after seeding, then cool at 0.3
C/min to À30
C, then at
À50
C to À150
C, and finally hold at À150
C for 10 min.
2. Plastic straws (IMV, L’Aigle, France).
3. Plugs for straws (IMV, L’Aigle, France), sealing powder or heat
sealer (see Note 2).
4. Pulled glass pipettes, denudation glass or plastic pipettes.
5. Tissue culture dishes (Falcon, Becton and Dickinson Co.,
USA).
6. Dissecting microscope.
7. Hotplate set at 37
C.
8. Forceps cooled in liquid nitrogen (optional—see Note 1).
9. Liquid nitrogen dewars, preferably at least two storage dewars
(see Note 3) and one for transporting samples to storage dewar
and cooling forceps, if used.
10. Scalpel.
11. Syringes and needle (optional—see Notes 4 and 5).
12. Heated water bath set at 30
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. Freezing solutions: 1.5 M 1,2-propanediol (PrOH) and 1.5 M
PrOH plus 0.2 M sucrose, both made up in PBS with protein
supplement.
18. Thawing and dilution solutions: 1.0 M PrOH plus 0.2 M
sucrose; 0.5 M PrOH plus 0.2 M sucrose; and 0.2 M sucrose,
all made up in PBS with protein supplement.
19. Hyaluronidase (optional—see Note 6).
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443
2.1 Slow Controlled
Rate Cooling
1. A controlled rate freezing machine (available from several
sources such as Planer plc, Middlesex, UK or Asymptote, Cambridge, UK) set to hold at 20
C, then cool at À2
C/min to
À7
C, allow for seeding (see Note 1), hold at À7
C for 10 min
after seeding, then cool at 0.3
C/min to À30
C, then at
À50
C to À150
C, and finally hold at À150
C for 10 min.
2. Plastic straws (IMV, L’Aigle, France).
3. Plugs for straws (IMV, L’Aigle, France), sealing powder or heat
sealer (see Note 2).
4. Pulled glass pipettes, denudation glass or plastic pipettes.
5. Tissue culture dishes (Falcon, Becton and Dickinson Co.,
USA).
6. Dissecting microscope.
7. Hotplate set at 37
C.
8. Forceps cooled in liquid nitrogen (optional—see Note 1).
9. Liquid nitrogen dewars, preferably at least two storage dewars
(see Note 3) and one for transporting samples to storage dewar
and cooling forceps, if used.
10. Scalpel.
11. Syringes and needle (optional—see Notes 4 and 5).
12. Heated water bath set at 30
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. Freezing solutions: 1.5 M 1,2-propanediol (PrOH) and 1.5 M
PrOH plus 0.2 M sucrose, both made up in PBS with protein
supplement.
18. Thawing and dilution solutions: 1.0 M PrOH plus 0.2 M
sucrose; 0.5 M PrOH plus 0.2 M sucrose; and 0.2 M sucrose,
all made up in PBS with protein supplement.
19. Hyaluronidase (optional—see Note 6).
Cryopreservation of Oocytes
443