cryopreservation of human oocytes and embryos

8/7/2019 Cryopreservation of human oocytes and embryos

1/16

R

RYOTOP SAFETY KITCCryotop Method

Protocol
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2/16

Vitrication

2

PART

Cryotop Safety Kit-Vitrication

No.0 Basic Solution (BS): 1 X 1.5ml vial (Only for Oocyte Vitrication)

No.1 Equilibration Solution (ES): 1 X 1.5ml vial

No.2 Vitrication Solution (VS): 2 X 1.5ml vials

4 Cryotops: 1 Cryotop stores up to 3 Oocytes or 3 Embryos as a recommendation.

2 Repro Plates: with 6 wells

Rack Cooling (Box for liquid nitrogen Ref. VT-CLB)

Pasteur pipette (Ref. MT-150)

Microscope (Turn o the heating plate.

Stopwatch or Timer (with count up function)

Liquid Nitrogen (Filter sterilization is available with Filter Teon Millipore.Tweezers

2 Micro pipettes: 2~20l

100~1000l

Cane

Storage tank

Materials Required

Use pasteur pipette that has a suitable internal diameter for Oocyte

(External diameter: 140m) or Embryo (External diameter: 140~200m)

to minimize Vitrication Solution and get a high survival rate after thawing.

CAUTION
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3/16

3

PART

Write necessary information about a patient on the label

of Cryotop (See Figure 1).

Fill 90% of a rack cooling with fresh liquid nitrogen.

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

(See Figure 2).

Note: For Oocyte Vitrication, take the cumulus cells o. Compare the width of perivitelline space with the thicknessof zona pellucida and record it (Ex. 1:1). It makes easy to

know the completing of the equilibration after immersing

in ES.

Figure 1

Figure 2

Oocyte

Preparation for Vitrication

Perivitelline Space

Zona Pellucida

2.

3.

4.

Bring BS, ES and VS to room temperature (25~27 ).1.

Vitrication Procedure

Cryotop

Vitrifcation

It is different procedures between Oocyte and Embryo for Equilibration.

Embryo EquilibrationCulture

OPU Oocyte Equilibration
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4/16

Vitrication

4

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 20l gently to the TOP ofBS with the

Oocyte moving micro pipette along the well and leave it for 3 minutes (See

Figure 4).


Add another ES 20l gently to the TOP ofBS as well and leave it for 3 minutes (See

Figure 4).


Add another ES 240 l gently to the TOP ofBS and leave it for 9 minutes (See Figure 4).

For Equilibration, the volume of Oocyte is required to be recovered completely.

Oocyte Equilibration is complete when the width of perivitelline space becomes

equal to the width before immersing to ES.

Oocyte Equilibration 1

Write BS, VS1 and VS2 on the rid of Repro Plate. Drop 20 l for BS and

300 l each for VS1 and VS2 on the plate with micro pipette (See Figure 3).

Immediately put the rid on the Repro Plate.

Oocyte Equilibration 2

Aspirate the Oocyte at the tip of the pasteur pipette. Transfer the Oocyte withminimal volume of medium from the culture dish to the BOTTOM ofBS

(20l).

Oocyte Equilibration

Figure 4

Figure 3

PART Equilibration
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5/16

5

Embryo Equilibration 3 For 12 or 15 minutes

Set up the stop watch (with count up function). Check the time with the stop 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

inltrating ES, which indicates that the Equilibration is complete.

Embryo Equilibration 2

Aspirate the Embryo at the tip of the pasteur pipette(See Figure 6).

Put the Embryo with minimal volume of medium to the TOP center ofES .

For Blastocyst Equilibration, wait for disappearing of the perivitelline space.

It is the best time when the diameter of Embryo reaches to 140180m for Blastocyst

vitrication .

Time taken for Equilibration depends on the size or the quality of the Embryo. High quality or

small Embryo doesnt take long time for Equilibration. For example, if a excellent grade Embryo

recovers completely for 8 minutes, the Equilibration step is nished. Stop Equilibration for 12

minutes for pronuclear stage and cleavage cell, for 15 minutes for morula stage and blastocyst if

the recovery of the volume of the Embryo can not be conrmed. It is enough for Equilibration.

Embryo Equilibration 1

Write ES,VS1 and VS2 on the rid of Repro Plate. Gently invert each vial ofES andVS twice to mix contents. Drop each solution 300l on the plate using micro

pipette (See Figure 5). Immediately put the rid on the Repro Plate.

Figure 5

Figure 6

Embryo Equilibration

CAUTION

Embryo


6/16

Vitrication

6

PART 4

It is the same procedure for Oocyte and Embryo. The following steps from 1 to 7 should be completed

between 60 and 90 seconds.

Vitrifcation 1

After the completion of Equilibration, aspirate the Oocyte (Embryo)

in ES at the tip of pasteur pipette (See Figure 7). Transfer the Oocyte

(Embryo) to the surface center ofVS1 with minimal volume ofES.

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 VS1 and blow it out again to

the outside of the well. Aspirate fresh VS1 into the pasteur pipette.

Vitrifcation 2 Within 1 minute

Aspirate the Oocyte (Embryo) in VS1 with the pasteur pipette and blow it out to VS1. Quickly stir ve times around the

Oocyte (Embryo). Repeat the aspirating, blowing out and stirring three times changing the positions in VS1 (See Figure

8). Displace the outer solution of the Oocyte (Embryo) to VS1 completely until the remaining ES visually disappears.

Vitrifcation 3 For 0.5 minutes

Blow out the remaining VS1 in the pasteur pipette to the outside of the well. Aspirate fresh VS2 into the pasteur pipete,

and then aspirate the Oocyte (Embryo) in VS1 at the tip of the pipette. Transfer the Oocyte (Embryo) to VS2 with minimal

volume ofVS1. Stir around the Oocyte (Embryo) changing positions twice with the pasteur pipette in VS2 (See Figure 8).

This step is completed when the outer Oocyte (Embryo) is displaced to VS perfectly and the at shrinking in cause ofdehydration is observed.

Vitrifcation 4

Place the Cryotop under a microscope (Logo should be up.) and

adjust the focus on the black mark of the Cryotop sheet (See

Figure 9).

Vitrication

Figure 7

Figure 8

Figure 9

Oocyte/Embryo


7/16

7

Vitrifcation 6

Check if the Oocyte (Embryo) is on the sheet with minimal volume ofVS2 under the microscope.

Plunge the Cryotop into fresh liquid nitrogen quickly.

Figure 11a: Good example

Step 1 Step 2 Step 3

Figure 11b: Bad example

Figure 12a: Good example Figure 12b: Bad example

Figure 10

Make a planar droplet by the black mark of Cryotopsheet.

Put the top of the pipette on the bottomend of the big VS drop.

Slide the pipette horizontally to outside,and make the VS drop lower.

Aspirate the excess VS, and minimizethe VS drop (not aspirating oocyte).

Make a steric droplet by the black mark of Cryotopsheet. The volume of VS2 is too much.

Vitrifcation 5

Aspirate the shrunk Oocyte (Embryo) in VS2 at the tip of the pasteur pipette

(See Figure 10). Place the Oocyte (Embryo) by the black part of Cryotop sheetwith minimal volume (less than 0.1l) ofVS2 (See Figure 11a and 11b). For

more than 2 Oocytes (Embryos), make 1 droplet for each (See Figure 12a and

12b).

Oocyte/Embryo

Removal of the excess VS on the sheet

After putting oocytes on the Cryotop sheet, the excess VS should be removed by aspirating using pipette.


8/16

Vitrication

8

Vitrifcation 7

Hold the straw cap with tweezers and insert the Cryotop from sheet end in liquid nitrogen. Then t the Cryotop with thecap by hands with screwing tightly in air (See Figure 13).

Vitrifcation 8

Check if the cap is xed with the Cryotop tightly. Put the Cryotop in a cane and store it in a storage tank.

Keep the Cryotop sheet in the liquid nitrogen until transferring to a storage tank.

In transferring the Cryotop to other storage tank, keep it in liquid nitrogen.

Do not expose of Cryotop in air until Thawing.

Figure 13

Twist it and make sure if the straw cap

ts tightly to the Cryotop.

Hold the straw cap with ngers and t it.Hold the straw cap with tweezers and insert

the Cryotop into it.

CAUTION


9/16

9


10/16

Thawing

10

PART

Cryotop Safety Kit -Thawing

No.1 Thawing Solution (TS): 1 X 4ml vial

No.2 Diluent Solution (DS): 1 X 1.5ml vial

No.3 Washing Solution (WS): 1 X 1.5ml vial

1 Petri Dish: 35mm for TS (Ref. FALCON 1008)

1 Repro Plate: with 6 wells

Rack Cooling (Box for liquid nitrogen Ref. VT-CLB)

Pasteur pipette (Ref. MT-150)

Microscope (Turn o the heating plate.)

Stopwatch or Timer (with count up function)

Liquid Nitrogen (Filter sterilization is available with Filter Teon Millipore.Tweezers

1 Micro pipettes: 100~1000 l

Cane

Storage tank

Materials Required

Use pasteur pipette that has a suitable internal diameter for Oocyte

(External diameter: 140m) or Embryo (External diameter: 140~200m)

to optimize the volume of the solutions for the best dilution condition to

get the highest survival rate.

CAUTION


11/16

11

Write DS,WS1 and WS2 on the rid of a Repro Plate.

Gently invert each vial of DS and WS twice to mix

contents. Drop 300l each for DS, WS1 and WS2 on the

Repro Plate with micro pipette. Place it on 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 ofTS twice to mix contents and pour the

full contents into the Petri Dish (See Figure 1).

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 (See Figure 2).

PART 2

Place the rack cooling by the stereo microscope.

Retrieve the cane which has the specic Cryotop, quickly immerse the cane in a rack cooling lled with fresh liquid

nitrogen. Retrieve the specic Cryotop from the cane in the liquid nitrogen. Check the information about the patient

on the label of Cryotop.

Warm TS vial (sealed) with a Petri Dish in an incubator to 37 (>1.5hours).

Preparation for Thawing

Figure 1

Figure 2

1.

2.

3.

4.

CAUTION

Pasteur pipette


12/16

Thawing

12

Oocyte/Embryo

PART 3 ThawingThawing 1

Carefully twist and remove the straw cap from

the Cryotop in liquid nitrogen (See Figure 3a).

Prop it against the corner of the rack cooling (See

Figure 3b).

Thawing 2

Be ready to use pasteur pipette keeping 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 minute

Quickly immerse Cryotop sheet into TS on the microscope stage. It should be within 1 second (See gure 4). 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 (See Figure 5).

Figure 3a Figure 3b

Figure 4

Figure 5

1


13/16

13

Oocyte/Embryo

Washing 1 For 5 minutes

3 minutes later, after immersing into DS, gently aspirate the

Oocyte (Embryo) in DS with the pasteur pipette. Also, aspirate

DS until the Oocyte (Embryo) reaches 2mm from the tip of the

pasteur pipette (See Figure 7).

Blow out only DS in the pasteur pipette into the BOTTOM center

of WS1 slowly (See Figure 8a), then gently place the Oocyte

(Embryo) on the bottom there (See Figure 8b). Leave it for 5

minutes. This is also for mostly gradual displacement from DS to

WS1.

Washing 2 For 1 minute

5 minutes later, after immersing into WS1, aspirate the Oocyte (Embryo)

with minimal volume of WS1 with pasteur pipette (See Figure 9) and

transfer it to the TOP center ofWS2. After the Oocyte (Embryo) free-falls

to the bottom ofWS2, do the same work again in WS2 (See Figure 10).

Washing 3

Transfer the Oocyte (Embryo) to a culture dish containing the appropriate culture medium. Incubate the Oocyte

(Embryo) in a 37 incubator to complete recovery.

For completing recovery : Oocytes for 2 hours and Embryos for 3 hours.

For your inquiry, email to: [email protected]

Figure 7

Figure 9

Figure 10

PART 5 Washing

PART 4 Dilution

Dilution For 3 minutes

Blow out only TS in the pasteur pipette into the BOTTOM

center ofDS slowly (See Figure 6a), then gently place the

Oocyte (Embryo) on the bottom of the TS layer (See Figure 6b).

Leave it for 3 minutes. This is for mostly gradual displacement

from TS to DS.

Figure 6a

Figure 8a

Figure 6b

Figure 8b

Oocyte/Embryo
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14/16

BS 20l

3min

1

min,37

TS

Before

After

LN2

3min

9min

0.5min

within1min

Cooling

(-23000/min)

Warming

(+42000/min)

1

2

3

1

2

Step1

Step2

Step3

WS

1

DS

1min

5min

3min

300l

300l

300l

WS2

1

2

20l

ES

Oocyt

e

20l

ES

ES

240l

300l

VS1

VS

2

300l
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itazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/


15/16

ES

300l

12or15min

Blastocyst

Before

After

4Cell

Embryo

W S

1

DS

1min

5min

3m

in

0.5min

within1min

1mi

n,37

TS

300l

300l

300l

LN2

Cooling

(-23000

/min)

Warming

(+42000/min)

1

2

3

1

2

300l

VS1

VS2

300l

WS2

Zygote 1

2
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itazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/http://www.kitazatoindia.com/


16/16

R
http://www.kitazatoindia.com/http://www.kitazatoindia.com/

cryopreservation of human oocytes and embryos

Documents