THAWING (KITAZATO)



Preparation for Thawing


Warm TS vial (sealed) wiht a Petri Dish in an incubator to 37℃(>1.5 Hours).
Bring DS and WS to room temprature (25~27℃).
Retrieve the cane which has the specific Cryotop, quickly immerse the cane in a Coolintg Rack filled with fress liquid nitrogen. Retrieve the specific Cryotop from the cane in the liquid nitrogen. Check the information about about the patient on the label of Cryotop.


White DS, WS1 and WS2 on the
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 stage and lid it.
Remove TS Vial and the Petri Dish from the incubator and place the petri dish on the microscope stage. Gently invert the vial of TS twice to mix contents and pour the full contents into the petri dish
Adjust the focus of the microscope to the petri dish with TS.Use pasteur pipette in order to focus easily on the center of the petri dish.


TS

fig4.2

DS

fig4.3

WS1

fig4.4

WS2

fig4.5

Cryotop® - Open System


Thawing 1

Carefully twist and remove the straw cap from the Cryotop in liquid nitrogen. Prop it against the corner of the Cooling Rack.


Thawing 2

Be ready to use pasteur pipette the Cryotop in liquid nitrogen. Set up the stop watch ( with count up function ). Check the time with the stop watch for the following steps




Thawing 3 - For 1 minutes

Quickly immerse Cryotop sheet into TS on the microscope stage. It should be within 1 second. Find the Oocyte (Embryo) adjusting the focus on the black mark of the Cryotop sheet. 1 minute after immersing into TS, gently aspirate the Oocyte (Embryo) with the pasteur pipette after dispensing it from the sheet. Aspirate the Oocyte (Embryo) even if it does not dispense from the sheet. Also aspirate TS until the Oocyte (Embryo) reaches 2mm from the tip of the pasteur pipette.


Cryotop® SC - Closed System


Thawing 1

Stand the CryotopSC on the Aluminum Block


Thawing 2

Cut the marking point with Straw Cutter.

Put the cutting blade at the black marking point. Turn the straw cap slowly to cut.

Thawing 3

Be ready to use pasteur pipette keeping the CryotopSC in liquid nitrogen. Set up the stop watch (with count up function). Check the time with the stop watch for the following steps.




Thawing 4

Insert the cut piece of the straw cap into the space between the CryotopSC and the Aluminum Block. This is to take out the CryotopSC easier.


Thawing 5 - for 1 minute

Quickly immerse the CryotopSC sheet into TS on the microscope stage by transferring it linearly. It should be within 1 second. Find the Oocyte (Embryo) adjusting the focus on the black mark of the Cryotop sheet. 1 minute after immersing into TS, gently aspirate the CryotopSC wiht the pasteur pipette after dispensing it from the sheet. Aspirate the Oocyte (Embryo) even if it does not dispende from the sheet. Also aspirate TS until the Oocyte (Embryo) reaches 2mm from the tip of the pasteur pipette.


Dilution


Dilution - for 3 minutes

Blow out only TS in the pasteur pipette into the BOTTOM center of DS slowly, Then gently place the Oocyte (Embryo) on the bottom of the TS layer.Leave it for 3 minutes. This is for mostly gradual displacement from TS to DS.




Washing


Washing 1- for 5 minutes

3 minutes lates, after immersing into DS, gently aspirate the Oocyte (Embryo) in DS wiht the pasteru pipette. Also, aspirate DS until the Oocyte (Embryo) reaches 2mm from the tio of the pasteur pipette.

Blow out only DS in the pasteur pipette into the BOTTOM center of WS1 slowly., then gently place the Oocyte (Embryo) on the bottom there. Leave it for 5 minutes. This is also for mostly gradual displacement from DS to WS1.


Washing


Washing 2- for 1 minutes

5 minutes lates, after immersing into WS1, aspirate the Oocyte (Embryo) with minimal volume of WS1 with pasteur pipette and transfer it to the TOP center of WS2. After the Oocyte (Embryo) free-falls to the bottom of WS2, do the same work again in WS2.




Washing


Washing 3

Transfer the Oocyte (Embryo) to a culture dish containg the appropriate culture medium. Incubate the Oocyte (Embryo) in a 37℃ inclubator to complete recovery.
Completion of recovery: Oocyte (Embryo) for 2 hourse for recommendation.