VITRIFICATION AND WARMING (KITAZATO)

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Figure 2

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Figure 4

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Preparation for Vitrification


Bring BS, ES and VS to room temprature (25-27℃).
Write necessary information about a patient on the handle/straw cap to cryotop.You can also label them.
[Cryotop] Fill 90% of cooling rack with fresh liquid nitrogen.
[Cryotop SC for closed system] Place aluminum block in cooling rack SC from the beginning. Then full with fresh liquid nitrogen until it covers the top of the aluminum block.
Remove the culture dish containing Oocyte or Embryo from the incubator. Check the quality of the Oocyte or the Embryo well with pasteur pipette under the microscope.
For Oocyte Vitrification, take the cumulus cells off.

Figure 5

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Figure 8

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Equilibration


Oocyte Equilibration 1

Write BS, VS1 and VS2 on the lid of Repro Plate. Drop 20µℓ for BS and 300µℓ each for VS1 and VS2 on the plate with micro pipette. Immediately put the lid on the Repro Plate.





Oocyte Equilibration 2

Aspirate the Oocyte at the top of the pasteur pipette. Transfer the Oocyte with minimal volume of medium from the culture dish to the BOTTOM of BS (20µℓ)


Oocyte Equilibration 3 - For 3 minutes

Set up the stop watch (with count up function). Check the time with the stop watch for the following steps. Add ES 20µℓ gently to the TOP of BS with the Oocyte moving micro pipette along the well and leave it from 3 minutes.


Oocyte Equilibration 4 - For 3 minutes

Add another ES 20µℓ gently to the TOP of BS as well and leave it for 3 minutes.


Oocyte Equilibration 5 - For 6 - 9 minutes

Add another ES 20µℓ gently to the TOP of BS and leave it for 6 - 9 minutes

For Equilibration, the volume of Oocyte is required to be recovered completely. Oocyte Equilibration is complete when the widht of perivitelline space becomes equal to the widht before immersing to ES.



Embryo Equilibration 1

Write ES, VS1 and VS2 on the lid of Repro Plate, Gently invert each vial of ES and VS twice to mix contents. Drop each solution 300 ul on the plate using micro pipette (See Figure 3-3). Immediately put the lid on the Repro Plate.


Embryo Equilibration 2

Aspirate the Embryo at the tip of the pasteur pipette (See Figure 3-4). Put the Embryo with minimal volume of medium to the TOP center of ES.




Embryo Equilibration 3 - For 10 - 15 minutes

Set up the stop watch (with count up function). Check the time with the strop watch for the following steps. The Embryo free-falls within 30 seconds. It spontaneously begins to shrink and then gradually returns to its original size with infiltrating ES, which indicates that the Equilibration is complete.


Vitrification

It is the same procedure for Oocyte and Embryo.

Vitrification 1

After the completion of Equilibration, aspirate the Oocyte (Embryo) in ES as the tip of pasteur pipette. Transfer the Oocyte (Embryo) to the surface center of VS1 with minimal volume of ES. Blow only the Oocyte (Embryo) out to VS1. To avoid getting the remaining ES in the pasteur pipette into the VS1, Blow out the ES to the outside of the well. Aspirate fresh VS! and blow it out again to the outside of the well. Aspirate fress VS1 into the pasteur pipette.




Vitrification 2 - Within 0.5 minute

Aspirate the Oocyte (Embryo) in VS1 with the pasteur pipette and blow it out to VS!. Quickly stir five times around the Oocyte (Embryo). Repeat the aspirating, Blowing out and stirring three time changing the positions in VS1. Displace the outer solution of the Oocyte the outer solution of the Oocyte (Embryo) to VS1 completely until the remaining ES visually disappears.


Vitrification 3 - Withing 0.5 minutes

Blow out the remaining VS1 in the pasteur pipette to the outside of the wall. Aspirate fresh VS2 into the Pasteur pipette, and then aspirate the Oocyte (Embryo) in VS1 at the tip of the pipette . Transfer the Oocyte (Embryo) to VS2 with minimal volume of VS1. Stir around the Oocyte (Embryo) changing position twice with the pasteur pipette in VS2. This step is completed when the outer Oocyte (Embryo) is displaced to VS perfectly and the flat shiring in cause of dehydration is observed.




Vitrification 4

Place the Cryotop under a microscope (logo should be up) and adjust the focus on the black mark of the Cryotop sheet.


Vitrification 5

Aspirate the Shrunk Oocyte (Embryo) in VS2 at the top of the pasteur pipette. Place the Oocyte (Embryo) by the black mark of Cryotop sheet with minimal volume (less the 0.1µℓ) of VS2. For more than 2 Oocytes (Embryos), make 1 droplet for each.



Removal of the excess VS on the sheet

After putting Oocytes (Embryos) on the Cryotop sheet, the excess VS should be remover by aspirating usgin pipette.