4 Notes
1. More than 90% of cumulus-enclosed immature oocytes survived this vitrification procedure and retained their competence
to undergo meiosis and fertilization [6]. Although oocyte
vitrification/warming reduces the percentage of embryo development, blastocyst stage embryos obtained from vitrified
oocytes were normal in terms of cell numbers and could
develop to term [6]. IVF-derived porcine zygotes survived
our vitrification protocol at rates over 95% without the reduction of their competence to develop to blastocysts [7–9].
2. Vitrification of immature porcine oocytes and zygotes either on
Cryotop
® sheets (Kitazato, Japan) in approximately 0.2 μL
vitrification solution or by dropping them into liquid nitrogen
in 1–2 μL aliquots of the vitrification solution were equally
effective [9, 12]. However, for the vitrification of porcine
blastocysts, Cryotop
® sheets were more feasible [9].
3. For the preparation of media, avoid the use of polypropylene
centrifuge tubes (such as Falcon 352097 or 352070). The
material of these tubes seems to interact with the vitrification
medium (i.e., dissolution of trehalose in these tubes took much
longer time than that in polystyrene tubes). Vitrification/
warming in media prepared in these tubes resulted in extremely
low survival rates.
4. Based on personal experience. The exact shelf life of each media
has not been determined.
5. PVP and bovine serum albumin are equally effective [12].
6. Sucrose and trehalose are equally effective [11].
7. Based on personal experience.
8. Base solutions of FDA are prepared with acetone. Acetone
dissolves polystyrene. Therefore, glassware or polypropylene
tubes (such as the Corning 430488 cryo-vial) must be used
to prepare FDA base solutions.
9. Any device to detect GFP fluorescence is feasible for this
staining.
10. The formation of white frost on the cooling metal surface
reduces the efficacy of vitrification. Dehumidification of the
room air slows this phenomenon.
11. Leaving a 3–4 cm rim prevents/reduces gas turbulence above
the proximity of the cooling surface which might affect the
speed of ice formation on the metal surface and thus cooling
efficacy. For the same reason, avoid placing the Styrofoam box
right in the ways of air ventilation.
12. The inner surface of the Petri dish lid can also be used.
466
Tamas Somfai and Kazuhiro Kikuchi
1. More than 90% of cumulus-enclosed immature oocytes survived this vitrification procedure and retained their competence
to undergo meiosis and fertilization [6]. Although oocyte
vitrification/warming reduces the percentage of embryo development, blastocyst stage embryos obtained from vitrified
oocytes were normal in terms of cell numbers and could
develop to term [6]. IVF-derived porcine zygotes survived
our vitrification protocol at rates over 95% without the reduction of their competence to develop to blastocysts [7–9].
2. Vitrification of immature porcine oocytes and zygotes either on
Cryotop
® sheets (Kitazato, Japan) in approximately 0.2 μL
vitrification solution or by dropping them into liquid nitrogen
in 1–2 μL aliquots of the vitrification solution were equally
effective [9, 12]. However, for the vitrification of porcine
blastocysts, Cryotop
® sheets were more feasible [9].
3. For the preparation of media, avoid the use of polypropylene
centrifuge tubes (such as Falcon 352097 or 352070). The
material of these tubes seems to interact with the vitrification
medium (i.e., dissolution of trehalose in these tubes took much
longer time than that in polystyrene tubes). Vitrification/
warming in media prepared in these tubes resulted in extremely
low survival rates.
4. Based on personal experience. The exact shelf life of each media
has not been determined.
5. PVP and bovine serum albumin are equally effective [12].
6. Sucrose and trehalose are equally effective [11].
7. Based on personal experience.
8. Base solutions of FDA are prepared with acetone. Acetone
dissolves polystyrene. Therefore, glassware or polypropylene
tubes (such as the Corning 430488 cryo-vial) must be used
to prepare FDA base solutions.
9. Any device to detect GFP fluorescence is feasible for this
staining.
10. The formation of white frost on the cooling metal surface
reduces the efficacy of vitrification. Dehumidification of the
room air slows this phenomenon.
11. Leaving a 3–4 cm rim prevents/reduces gas turbulence above
the proximity of the cooling surface which might affect the
speed of ice formation on the metal surface and thus cooling
efficacy. For the same reason, avoid placing the Styrofoam box
right in the ways of air ventilation.
12. The inner surface of the Petri dish lid can also be used.
466
Tamas Somfai and Kazuhiro Kikuchi