Warming Thawing Protocol

Materials

  • Cryotech Warmig Kit
    • Warming Solution (TS) :1 vial of 1.8mℓ
    • Diluent Solution (DS) :1 vial of 0.5mℓ
    • Washing Solution (WS) :1 vial of 1.0mℓ
    • 1 Warming Plate with 4 wells
  • Microscope (Turn off the heating plate)
  • Stop watch (With count up function)
  • Tweezers
  • Micro pipette for 300μℓ

Preparation

Warming Plate

Fig.7.Preparation Warming Plate

  1. 1
    Place the Warming Plate and the TS vial (with closed cap) in the incubator at 37℃ > 3 hours before warming (overnight storage is recommended).
  2. 2
    Bring the DS and the WS vials to room temperature (25 to 27℃) at least 1 hour before warming.
  3. 3
    Prepare fresh liquid nitrogen.
  4. 4
    Take the patient’s cane out of a liquid nitrogen tank, and take off the cover cap and prop up the Cryotec against inside wall the cooling rack in liquid nitrogen.
  5. 5
    Take the Warm Plate out of the incubator, and fill the second well with 300μℓ of the DS (Fig.7).
Warming (1min)

Figs.8.Warming Procedure (Step 1-3)

  1. 1
    Take the TS vial out of the incubator, and expel all of the solution out of it into the TS well (1.8ml, Figs. 8, Step1/①)
  2. 2
    Quickly (within 1 sec) put the Cryotec from liquid nitrogen into the TS well (Figs. 8, Step1/②). Start counting up by the stop watch for 1 min.
  3. 10
    The oocyte/embryo releases from the Cryotec sheet by itself, and begins to float.

Vitrification1 (30-40sec)

1
2
3
Figs.9.Gradual Replacement of Solutiond (Dilution)
  1. 1
    Aspirate the oocyte/embryo first, followed by 3mm of the TS into the pipette (Figs. 9， 1).Aspirate the oocyte/embryo first, followed by 3mm of the TS into the pipette (Figs. 9， 1).
  2. 2
    Introduce the TS to the bottom of the DS well (Figs. 9, 2), then expel the oocyte/embryo slowly to
  3. 3
    the bottom of TS layer in DS well (Figs. 9, 3), and wait for 3 min (Fig. 8, Step 2/①).
  4. 4
    While waiting, fill the WS1 and the WS2 well with 300μℓ each of ws Solution (Figs. 8, Step 3/①).

Washing (5min)

1
2
3
Figs.10.Gradual Replacement of Solutiond (Washing 1 Step)
  1. 1
    Aspirate the oocyte/embryo followed by 3mm of the DS into the pipette (Figs. 10，1).
  2. 2
    Introduce the DS to the bottom of the WS1 (Figs. 10, 2), and expel the oocyte/embryo slowly to the bottom of the DS layer in WS1 well (Figs. 10.3). Observe the shape of the oocyte/embryo and memorize it. Turn off the light, and wait for more than 3 min.
  3. 3
    After 3 min, compare the shape of the oocyte/embryo to the one memorized. Give ａ survival judgment if the shrinkage of the oocyte is recovered.
  4. 4
    Wait for 5 min in total (Figs. 8, Step3/②).

Washing2 (1min)

  1. 1
    Aspirate the oocyte/embryo with minimal volume of the WS1 .
  2. 2
    Put the oocyte/embryo on the surface of the WS2 well (Fig. 8, Step3/③).
  3. 3
    After the oocyte/embryo sinks to the bottom, aspirate and place it on the surface of a different location with in W2. Put the oocyte/embryo into the droplet of the culture media until ICSI or ET is performed.
  4. 4
    (two to four hours culture for ICSI, and 3 hours for blastocyst transfer are recommended)

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