REPROLIFE

Warming

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. 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. Bring the DS and the WS vials to room temperature (25 to 27℃) at least 1 hour before warming.
  3. Prepare fresh liquid nitrogen.
  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. 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. 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. 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. 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. Aspirate the oocyte/embryo first, followed by 3mm of the TS into the pipette (Figs. 9, 1).
  2. Introduce the TS to the bottom of the DS well (Figs. 9, 2), then expel the oocyte/embryo slowly to
  3. the bottom of TS layer in DS well (Figs. 9, 3), and wait for 3 min (Fig. 8, Step 2/①).
  4. While waiting, fill the WS1 and the WS2 well with 300μℓ each of ws Solution (Figs. 8, Step 3/①).

Washing (5min)

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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. 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. 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. Wait for 5 min in total (Figs. 8, Step3/②).

Washing2 (1min)

  1. Aspirate the oocyte/embryo with minimal volume of the WS1 .
  2. Put the oocyte/embryo on the surface of the WS2 well (Fig. 8, Step3/③).
  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. (two to four hours culture for ICSI, and 3 hours for blastocyst transfer are recommended)