Warming Thawing Protocol
- 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)
- Micro pipette for 300μℓ
- Place the Warming Plate and the TS vial (with closed cap) in the incubator at 37℃ > 3 hours before warming (overnight storage is recommended).
- Bring the DS and the WS vials to room temperature (25 to 27℃) at least 1 hour before warming.
- Prepare fresh liquid nitrogen.
- 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.
- Take the Warm Plate out of the incubator, and fill the second well with 300μℓ of the DS (Fig.7).
- 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)
- 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.
- The oocyte/embryo releases from the Cryotec sheet by itself, and begins to float.
Figs. 9. Gradual Replacement of Solutiond (Dilution)
- Aspirate the oocyte/embryo first, followed by 3mm of the TS into the pipette (Figs. 9， 1).
- Introduce the TS to the bottom of the DS well (Figs. 9, 2), then expel the oocyte/embryo slowly to
- the bottom of TS layer in DS well (Figs. 9, 3), and wait for 3 min (Fig. 8, Step 2/①).
- While waiting, fill the WS1 and the WS2 well with 300μℓ each of ws Solution (Figs. 8, Step 3/①).
Figs. 10. Gradual Replacement of Solutiond (Washing 1 Step)
- 1.Aspirate the oocyte/embryo followed by 3mm of the DS into the pipette (Figs. 10，1).
- 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.
- 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.
- Wait for 5 min in total (Figs. 8, Step3/②).
- Aspirate the oocyte/embryo with minimal volume of the WS1 .
- Put the oocyte/embryo on the surface of the WS2 well (Fig. 8, Step3/③).
- 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.
- (two to four hours culture for ICSI, and 3 hours for blastocyst transfer are recommended)