Frequently Asked Questions
As TS and plates should be used at 37°C, place them in a warmer, incubator, etc., at least 2 hours before the start of thawing operation. They can be placed there overnight. Use other reagents, DS and WS, at room temperature (25°C-27°C).
It can be used up to twice for the same patient in compliance with the protocol and with attention to the maintenance of appropriate temperature (37°C for thawing plate and TS, 25°C-27°C for DS and WS). At that time, start the second thawing while waiting for the WS1 step of the first thawing.
It is limited to the same patient, but if the plate is used for the second thawing immediately after the WS1 step of the first thawing, temperature will drop. Therefore, pay particular attention to the temperature control when it is used for the second thawing.
Although the inside of the bag is sterilized, dust, etc., may adhere to the outside of the bag after opening the box. Therefore, we recommend that the plate be taken out of the bag when it is placed in the sterile area such as an incubator.
The temperature of TS should be as high as possible in order to maximize the heating rate and to quickly pass through the critical temperature range for ice crystal formation. As the maximum temperature used for oocytes/embryos is 37°C, TS should be heated to 37°C until immediately before use.
As HEPES is added to TS, we recommend the use of an incubator or warmer without CO2. When using the CO2 incubator, tighten the vial of TS again and wrap it with parafilm before placement. If you are concerned, it is safe to put each vial in a larger conical tube, etc., and close its cap for preparation.
Unused TS can be used without any problem. Stop heating and store it again in the refrigerator.
Only in case of emergency, warm TS in hot water at 40°C for 3 minutes for thawing. Store the thawing plate in a warmer at 37°C until immediately before use.
It can be used up to twice for the same patient in compliance with the protocol and with attention to the maintenance of appropriate temperature (37°C for thawing plate and TS, 25°C-27°C for DS and WS). At that time, start the second thawing while waiting for the WS1 step of the first thawing.
Use the one with a size suitable for the oocyte/embryo to be thawed. We recommend 140 to 150 μm for oocytes/cleavage-stage embryos and 200 to 250 μm for blastocysts.
The temperature of Cryotec rises from the moment it is removed from liquid nitrogen. Quickly insert it into TS within 1 second.
If liquid nitrogen remains in the Cryotec sheet, it will form bubbles in the TS solution. When withdrawing Cryotec from liquid nitrogen, take it out vertically from the liquid level as much as possible. When inserting it in TS, bubble formation can be reduced by inserting the sheet at an incidence angle of 20° to 30° along the curve of the TS well.
The oocyte/embryo is frozen in a state of dehydration until the amount of water in the cell reaches about 50%. Therefore, it usually floats immediately after thawing due to the difference in specific gravity with TS.
If Cryotec is removed, the cooled item will be moved in warm liquid, and the temperature of TS will drop faster. In addition, the moment of immersion in TS is the most stressful time for oocytes/embryos, so it is necessary to wait without moving them as much as possible to avoid further stress. Therefore, Cryotec should remain still in TS for 1 minute even if the oocytes/embryos have floated.
If the oocyte/embryo still adheres to the sheet after one minute of immersion in TS, (1) gently shake the Cryotec horizontally until the oocyte/embryo comes off naturally. If it still does not come off, (2) slowly spray TS and peel off from the sheet. We recommend you try the above two methods because oocytes/embryos may be directly contacted if they are directly removed with the pipette.
As the droplet is not minimized at the time of loading, oocytes/embryos may be detached from the sheet immediately when Cryotec is immersed in TS. However, if the oocyte/embryo was certainly loaded on the sheet, it must be in TS solution for sure. At 1 minute after immersion in TS, confirm that there is no oocyte/embryo on the sheet, remove Cryotec, and take time to look for the surface or edge in the TS well.
On a rare occasion, an embryo may float in DS particularly when the blastocyst is in good condition and volume recovery is fast. It is due to the change in the specific gravity inside and outside of the embryo; in other words, recovery has been smooth. Therefore, there is no need to place it at the bottom of the well again.
The required amount is 3 mm, so there is no need to add more from the top.
The time for each step of thawing (1 minute for TS, 3 minutes for DS, 5 minutes for WS1) is the minimum time for immersion. Even if embryo recovery is already observed, be sure to immerse in each reagent within the specified time.
If an oocyte/embryo is dead, volume recovery cannot occur because the function of cell membrane is lost and water cannot flow in. Therefore, if volume recovery is seen in WS1, survival can be simply determined.
They have already been washed in WS2, and there is almost no difference in osmotic pressure or composition from the culture solution, so there is no problem even if it is mixed. Therefore, it is left to each institution whether or not to wash.
Culture for 2 hours after thawing is recommended. Two hours of culture is not necessary if the spindle is confirmed in less than 2 hours.
As we consider that the in vivo is a more suitable environment for embryos than the in vitro, we recommend that embryo transfer be performed as soon as survival is confirmed. We usually inform that it should be after about 1 hour of culture after thawing. However, please follow the clinic's policy.
