6. Prior to warming the straws, prepare dishes containing a
0.5–1 mL droplet each of 1.0 M PrOH plus 0.2 M sucrose,
0.5 M PrOH plus 0.2 M sucrose, 0.2 M sucrose, and PBS with
protein supplement. Label each droplet.
7. Wearing suitable protective equipment, warm the straws by
holding them in air for 30 s, hold either end of the straw not
the area containing the oocytes. Then place the straws in the
water bath set at 30
C for 30 s or until the ice has just melted.
8. Wipe the straw dry. The plug, if used, should be removed from
the straw or the ends of the straw cut with a scalpel and the
contents then expelled (see Note 5) into the dish containing
1 M PrOH plus 0.2 M sucrose.
9. The oocytes should remain in this solution for 5 min at room
temperature before being moved into the droplet of 0.5 M
PrOH plus 0.2 M sucrose again for 5 min at room temperature.
10. The oocytes are moved into 0.2 M sucrose for 10 min at room
temperature.
11. The oocytes are then placed in the droplet of PBS for 20 min,
10 min at room temperature and 10 min at 37
C on a
hotplate.
12. The oocytes should then be cultured (2–3 h for human, 30 min
for mouse) within an incubator in a suitable culture medium to
allow recovery prior to attempted fertilization (see Note 13).
3.2 Vitrification
Vitrification is advantageous over slow controlled rate cooling in
that neither expensive cooling machines nor special laboratory
environment are required. However, the need for high concentrations of CPA means that the times stated for CPA exposure must be
strictly adhered to and manipulation of oocytes in these highly
viscous solutions is technically demanding. Different CPA mixtures
and minimum volume VS have been proven to be effective for
oocyte VS. Toxicity of VS solution can be reduced by combination
of decreased concentration of permeating CPA, which can be balanced by increasing concentrations of non-permeating agents like
polymers and sugars [53, 67–69]. Accurately performed VS (see
Fig. 2) can result in oocytes with similar to unfrozen oocytes postwarming morphological recovery, embryo development, and life
birth in human. The VS method described using VSDP has resulted
in high blastocyst formation [70] and live births [71] in mice,
although the technique is prone to variability [48, 67]. The CPA
mixture and cryopreservation vessel could be modified, but the
basic techniques described are applicable to all VS protocols.
1. Straws should be prepared by pushing the cotton plug approximately one third of the way down the straw. Using a needle and
syringe, inject a column of sucrose solution, via what was the
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Victoria Keros and Barry J. Fuller
0.5–1 mL droplet each of 1.0 M PrOH plus 0.2 M sucrose,
0.5 M PrOH plus 0.2 M sucrose, 0.2 M sucrose, and PBS with
protein supplement. Label each droplet.
7. Wearing suitable protective equipment, warm the straws by
holding them in air for 30 s, hold either end of the straw not
the area containing the oocytes. Then place the straws in the
water bath set at 30
C for 30 s or until the ice has just melted.
8. Wipe the straw dry. The plug, if used, should be removed from
the straw or the ends of the straw cut with a scalpel and the
contents then expelled (see Note 5) into the dish containing
1 M PrOH plus 0.2 M sucrose.
9. The oocytes should remain in this solution for 5 min at room
temperature before being moved into the droplet of 0.5 M
PrOH plus 0.2 M sucrose again for 5 min at room temperature.
10. The oocytes are moved into 0.2 M sucrose for 10 min at room
temperature.
11. The oocytes are then placed in the droplet of PBS for 20 min,
10 min at room temperature and 10 min at 37
C on a
hotplate.
12. The oocytes should then be cultured (2–3 h for human, 30 min
for mouse) within an incubator in a suitable culture medium to
allow recovery prior to attempted fertilization (see Note 13).
3.2 Vitrification
Vitrification is advantageous over slow controlled rate cooling in
that neither expensive cooling machines nor special laboratory
environment are required. However, the need for high concentrations of CPA means that the times stated for CPA exposure must be
strictly adhered to and manipulation of oocytes in these highly
viscous solutions is technically demanding. Different CPA mixtures
and minimum volume VS have been proven to be effective for
oocyte VS. Toxicity of VS solution can be reduced by combination
of decreased concentration of permeating CPA, which can be balanced by increasing concentrations of non-permeating agents like
polymers and sugars [53, 67–69]. Accurately performed VS (see
Fig. 2) can result in oocytes with similar to unfrozen oocytes postwarming morphological recovery, embryo development, and life
birth in human. The VS method described using VSDP has resulted
in high blastocyst formation [70] and live births [71] in mice,
although the technique is prone to variability [48, 67]. The CPA
mixture and cryopreservation vessel could be modified, but the
basic techniques described are applicable to all VS protocols.
1. Straws should be prepared by pushing the cotton plug approximately one third of the way down the straw. Using a needle and
syringe, inject a column of sucrose solution, via what was the
446
Victoria Keros and Barry J. Fuller