18. Dilution solution consisting of 1 M sucrose made up in PBS
containing protein supplement.
19. Hyaluronidase (optional—see Note 6).
3 Methods
3.1 Slow Controlled
Rate Cooling
The slow controlled rate cooling method described uses a mixture
of the permeating cryoprotectant PrOH and non-permeating
sucrose. It has been shown that 0.3 M sucrose yields better survival
than 0.2 M sucrose in human oocytes [63, 64]. This is thought to
be largely due to greater dehydration of the cells prior to freezing.
Optimal exposure time to PrOH plus 0.2 M sucrose is 5–10 min,
whereas optimal exposure time to PrOH plus 0.3 M sucrose is yet
to be determined. However, an exposure time of 2 min will give a
level of dehydration equivalent to that achieved with 5 min exposure to PrOH plus 0.2 M sucrose [45]. A further potential modification to the method described below is the choice of media in
which to dilute the CPAs. Studies have also shown a medium in
which some of the sodium has been replaced by choline to be
preferable to PBS [37, 65, 66].
1. Straws should be loaded with a column of 1.5 M PrOH plus
0.2 M sucrose and left at room temperature (20 Æ 2
C) until
required (see Note 4).
2. Place one 0.5 mL droplet each of PBS plus protein supplement,
1.5 M PrOH and 1.5 M PrOH plus 0.2 M sucrose in a tissue
culture dish. Label each droplet.
3. No more than five oocytes (see Note 11) should be placed in
the droplet of PBS (cumulus cells may be removed, see Note 6)
and then placed in the droplet of 1.5 M PrOH for 10 min at
room temperature. The oocytes are then moved to the droplet
of 1.5 M PrOH plus 0.2 M sucrose for 5 min at room
temperature.
4. The oocytes are then loaded into the prepared straws within the
column of 1.5 M PrOH plus 0.2 M sucrose and the straw
sealed (see Note 2).
5. The straws should be placed within a freezing machine set at
20
C. The machine should be cooled to À7
C at À2
C/min.
Having reached this temperature, ice nucleation should then be
initiated in the solution containing the oocytes (see Note 1).
The straws are then replaced in the cooling machine and the
cooling regime resumed with the temperature being held at
À7
C for 10 min to allow dissipation of the heat of crystallization. Once the cooling protocol has been completed and the
samples are being held at À150
C, wearing appropriate protective equipment, the straws are placed in a liquid nitrogen
storage dewar (see Note 12).
Cryopreservation of Oocytes
445
containing protein supplement.
19. Hyaluronidase (optional—see Note 6).
3 Methods
3.1 Slow Controlled
Rate Cooling
The slow controlled rate cooling method described uses a mixture
of the permeating cryoprotectant PrOH and non-permeating
sucrose. It has been shown that 0.3 M sucrose yields better survival
than 0.2 M sucrose in human oocytes [63, 64]. This is thought to
be largely due to greater dehydration of the cells prior to freezing.
Optimal exposure time to PrOH plus 0.2 M sucrose is 5–10 min,
whereas optimal exposure time to PrOH plus 0.3 M sucrose is yet
to be determined. However, an exposure time of 2 min will give a
level of dehydration equivalent to that achieved with 5 min exposure to PrOH plus 0.2 M sucrose [45]. A further potential modification to the method described below is the choice of media in
which to dilute the CPAs. Studies have also shown a medium in
which some of the sodium has been replaced by choline to be
preferable to PBS [37, 65, 66].
1. Straws should be loaded with a column of 1.5 M PrOH plus
0.2 M sucrose and left at room temperature (20 Æ 2
C) until
required (see Note 4).
2. Place one 0.5 mL droplet each of PBS plus protein supplement,
1.5 M PrOH and 1.5 M PrOH plus 0.2 M sucrose in a tissue
culture dish. Label each droplet.
3. No more than five oocytes (see Note 11) should be placed in
the droplet of PBS (cumulus cells may be removed, see Note 6)
and then placed in the droplet of 1.5 M PrOH for 10 min at
room temperature. The oocytes are then moved to the droplet
of 1.5 M PrOH plus 0.2 M sucrose for 5 min at room
temperature.
4. The oocytes are then loaded into the prepared straws within the
column of 1.5 M PrOH plus 0.2 M sucrose and the straw
sealed (see Note 2).
5. The straws should be placed within a freezing machine set at
20
C. The machine should be cooled to À7
C at À2
C/min.
Having reached this temperature, ice nucleation should then be
initiated in the solution containing the oocytes (see Note 1).
The straws are then replaced in the cooling machine and the
cooling regime resumed with the temperature being held at
À7
C for 10 min to allow dissipation of the heat of crystallization. Once the cooling protocol has been completed and the
samples are being held at À150
C, wearing appropriate protective equipment, the straws are placed in a liquid nitrogen
storage dewar (see Note 12).
Cryopreservation of Oocytes
445