REPROLIFE

Vitrification

Vitrification Protocol

Materials

  • Cryotech Vitrification Kit
     Equilibration Solution (ES) : 1 vial of 1.0mℓ
     Vitrification Solution (VS) : 2 vials of 1. 0mℓ
  • Microscope (Turn off the heating plate)
  • Stop watch (with count up function)
  • Tweezers
  • Scissors
  • Micro pipette for 300μℓ

Preparation

Bring ES and VS vials to room temperature (25℃ to 27℃) at least 1 hour before vitrification.

Equilibration (12-15min)

Vitri Plate
Fig. 1. Preparation of Each Solution
Fig. 2. ES Equilibration
  1. Fill the wells of Vitri Plate with 300μℓ of ES and 300μℓ of VS (Fig. 1). Put the lid on the Vitri Plate immediately.
  2. Observe the oocyte/embryo well and remember each perivitelline space to know the full recovery volume of it during ES.
  3. Aspirate the oocyte/embryo at the tip of pasteur pipette.
  4. Put the oocyte/embryo with a small amount of medium on the surface of the ES (Fig.2). Start counting up by the stop watch.
  5. Cover the Vitri Plate with the lid, and wait for the full reocvery from the shrinkage.
  6. While waiting, open a “Cryotec package” with scissors. Write the information of the oocyte/embryo on the “handle” of Cryotec, and put it in the groove of the Vitri Plate (Fig.3).
Fig. 3. Cryotec on Vitri Plate
  1. Prepare fresh liquid nitrogen, and put a cover cap in it
  2. When the oocyte/embryo’s volume is completely recovered, ES equilibration ends.

If you can’t confirm the recovery completely, the limit time of ES equilibration is 15 min for the oocyte and blastocyst (160-220 μm in diameter), and 12 min for 4 – 8 cells embryo.

Vitrification1 (30-40sec)

VS1

Step 1
Step 2
Step 3
Step 4

Figs. 4. VS1 Washing of the Oocyte/Embryo (Step 1 – 4)

  1. Aspirate the oocyte/embryo with a small amount of ES at the tip of the pipette・.
  2. Place the oocyte/embryo in mid depth of VS1 (Step 1).
  3. Rinse out the pipette expelling out of wells the ES left inside (Step 2/①), aspirate VS1 from the edge of the well (Step 2/②) and immediately expel it.  (rinse out inside the pipette, Step 2/③)
  4. The oocyte/embryo will float immediately to the surface of VS1 (Step 2/④). After aspirating fresh vs from the edge, aspirate the oocyte/embryo at the tip of the pipette.
  5. Place now the oocyte/embryo again to the bottom of VS1 (Step3 ).
  6. The oocyte/embryo will rise to mid depth and will stop (End of VS1 equilibration, Step 4).
  7. Rinse out the pipette expelling the VS1 left inside, aspiratnig and expelling fresh VS2. Aspirate VS2, followed by oocyte/embryo in VS1 at the tip of the pipette

Vitrification2 (10-20sec)

VS2

Step 5
Step 6
Step 7
Step 8

Figs. 5. VS2 Washing of the Oocyte/Embryo (Step 5 – 8)

  1. Place the oocyte/embryo to the mid depth of the VS2 (Step 5).
  2. Rinse out the pipette with fresh VS2 (Step 6/①②③)
  3. Aspirate fresh VS2 from the edge of the well, mix the solution around the oocyte/embryo, and observe the complete shrinkage of the oocyte/embryo (Step 7).
  4. Take the oocyte/embryo at the tip of the pipette (Step 8).
  5. Place the oocyte/embryo near the black mark on the Cryotec sheet with small volume of the VS2. (One oocyte/embryo per droplet is recommended:Fig.6).
  6. Immediately submerge and gently stir the Cryotec in fresh liquid nitrogen.
  7. Put the cover cap on the Cryotec inside the liquid nitrogen.
Figd. 6. Oocyte/Embryo on the Cryotec

Note 1

Use the optimum size pasteur pipette.

  • 140-150 μm for oocyte and cleavage stage embryo.
  • 160~220 μm for blastocyst.(Depends on the size of the blastocysts)

Note 2

Best timing for vitrification of blastocyst: the size (diameter) should be between 160~220 μm for perfect survival after vitirfication.