Vitrification Protocol
for Oocytes / Embryos

2

Vitriﬁcation Protocol

Vitriﬁcation Procedure
Oocytes
Embryos

ES Equilibration (3 steps)（Artiﬁcial Shrinkage）

ES Equilibration (1 step)

VS Equilibration

Vitriﬁcation

Cryo Storage

PART1 Materials Required
1. Vitriﬁcation Media VT601 / VT621 [Ref. 91101 / 91207]
＊1

No.0

BS (Basic Solution) 1.5mL vial ×1

No.2

VS (Vitriﬁcation Solution) 1.5mL vial ×2

No.1

ES (Equilibration Solution) 1.5mL vial ×1＊1＊2

For oocytes vitriﬁcation
For preparation of blastocysts vitriﬁcation※Equilibrate all the solutions to room temperature before use

2．Shrinkage Solution VT525-4 [Ref. 91366]＊2

3．Cryotop (1 Cryotop is for maximum 4 oocytes/embryos)

4．Oocyte Cryo Plate [Ref. 83061] (for oocytes) / Repro Plate (for embryos)

5．Pipette for handling oocytes / embryos (hereinafter referred to as “pipette”)
6．Timer (with count up function)
7．Liquid nitrogen

8．Cooling Rack [Ref. 84014] for liquid nitrogen container
9．Forceps

10．Micro pipettes: 20μL (for oocytes), 300μL
11．Cryo storage equipment (cane, etc.)

12．Stereo microscope (stage must be turned oﬀ)

TIPS
Use pipette that has a suitable internal diameter for oocytes or embryos for better handling. (For oocytes: about120-140μm, for cleavage stage embryos: about 150-180μm ,and 180-250μm for blastocysts. )All vitriﬁcation procedures are performed at room temperature.

Vitrification

3

Vitriﬁcation Protocol

PART2 Preparation before Blastocysts Vitriﬁcation

In the case of blastocysts reaching the expanded stage (Grade 4 or higher according to the Gardner classiﬁcation),
there is an increased risk of insuﬃcient replacement of water with cryoprotectant. Therefore, we recommend performing
shrinkage artiﬁcially using Shrinkage Solution (VT525-4) prior to ES equilibration.

Shrinkage Solution 300µL

Preparation

Bring Shrinkage Solution to room temperature and dispense 300 μL on Repro Plate, cover with a lid andleave it before use.

ES equilibration procedure will follow when shrinkage is conﬁrmed, so preparation for ES equilibration on
P.5 (Prepare one ES well and two VS wells, respectively 300μL) should be made at the same time.

STEP1Blastocysts Shrinkage

Transfer the blastocysts into the Shrinkage Solution and conduct pipetting to stimulate shrinkage .
After conﬁrming the shrinkage of the blastocysts, accelerate ES equilibration on P.5.

※ Proceed to ES equilibration after 2 minutes if the shrinkage of the blastocysts cannot be conﬁrmed.

Information
Applicability Guide for Shrinkage Solution
Developmental stages of blastocyst

Grade 1

Early blastocyst

Grade 2

blastocyst

Grade 3Full blastocyst

Grade 4

Expanded blastocyst

Grade 5

Hatching blastocyst

Grade 6

Hatched blastocyst

Shrinkage with a Shrinkage Solution (VT525-4) is recommended
from an d afte r the e xpan di n g blas toc ys t s tag e4Vitrification

PART3 ES Equilibration

Vitriﬁcation Protocol

ES equilibration procedure is diﬀerent for oocytes and embryos. Refer to this page for embryos and P.6 for oocytes.
(The subsequent procedures are the same between the two.)

Embryos ES Equilibration
ES 300µL

1

VS1 300µL

2VS2 300µL

ES

315 minPreparation

STEP1

Prepare one ES well and two VS wells, respectively 300μL on a Repro Plate.
Cover the Repro Plate until ready to use.

Transfer of Embryos to ES

Aspirate the embryos near the tip of the pipette and gently place itwith minimal volume of culture medium to the surface center of ES
to avoid dilution.

STEP2Fig.1

ES Equilibration

Set the timer and leave the embryos still for 15 min.

The embryos placed on the surface will sink as they shrink in about 30 seconds.

to P.7

Vitrification

5

Vitriﬁcation Protocol
The ES equilibration procedure is diﬀerent for oocytes and embryos. Refer to this page for oocytes andP.5 for embryos. (The subsequent procedures are the same between the two.)

Oocytes ES Equilibration
1. BS 20μL 2. ES 20μL
3min①②

4. ES 300μL ③ 9min

3min

3. ES 20μL

1 23 4Merge ES to BS(First time)Merge ES to BS(Second time)

1. BS 20μL 2. ES 20μL
3min①

1. BS 20μL 2. ES 20μL
3min①②②Oocytes

3min

3. ES 20μL

3 min

Preparation

13min

3. ES 20μL

3 min

1BS 20µL

On Oocytes Cryo Plate, dispense 20 μL drop of BS in area
300 μL of ES in area

, 300 μL of VS in area

respectively. Keep the lid on until use (Fig. 1).

ESand,

9 min

ES 300µL

1. BS 20μL 2. ES 20μL
3min①②

4. ES 300μL ③ 9min

3min

3. ES 20μL

1 23 4

VS1 300µL

STEP1
STEP2

STEP4

Transfer the oocytes from the culture medium to the BS drop.
OocyteMerging ES (First time)

Dispense 20 μL of ES drop on the right side of the BS drop, and

1. BS 20μL 2. ES 20μL
3min①

merge the ES to the BS drop using the chip of the pipette, then

②

3min

3. ES 20μL

ES 20µL
Fig.2

1OocyteMerging ES (Second time)

Dispense 20 µL drop of ES below the mixed drop. Merge the new
ES drop to it with the chip, and leave it still for 3 min (Fig. 3).

1. BS 20μL 2. ES 20μL
3min①②3min3. ES 20μL

1ES 20µL

Transfer of Oocytes to ES
and leave it still for 9 min (Fig. 4).

TIP

Although it may be diﬃcult to conﬁrm the recovery of oocytes depending on the angle of observation, STEP4 iscompleted within 9 minutes even in such cases.

Merging the drops allows gentle osmolality change, so leave the oocytes untouched.

Vitrification

Fig.3

Transfer the oocytes to the surface of the dispensed ES in area

9 min.

6Fig.1

Transfer of Oocytes to BS

leave it still for 3 min (Fig. 2).

STEP3

VS2 300µL

Fig.4

Vitriﬁcation Protocol

PART4 VS Equilibration

From this page onwards, the procedures for oocytes and embryos are the same.
Prepare Cryotop and liquid nitrogen before starting the following steps.
VS1（STEP3）
1VS2（STEP4）

2

1

3

2×5

STEP1×560 - 90 sec

Transfer of Oocytes/Embryos to VS1

After completion of ES equilibration, aspirate the oocytes/embryos withminimal volume of ES at the tip of the pipette (Fig. 1).

Transfer the aspirated oocytes /embryos to the surface of VS1 and set
the timer.

STEP2

Fig.1

Washing Pipette

Expel the remaining ES in the pipette to the outside the well, andwash the pipette by aspirating and expelling suﬃcient volume of VS(Fig. 2 and 3).

STEP3

VS1 Equilibration

ESVS

Fig.2

Aspirate the oocytes/embryos and perform steps the below ① and ② in three positions in the VS1.

VS
Fig.3

① Expel the oocytes/embryos from the pipette to the VS.

② Stir around the oocytes/embryos 5 times gently for 5 seconds.

The oocytes/embryos are equilibrated and dehydrated by performing steps ① and ② in three diﬀerentpositions.

STEP4

VS2 Equilibration

Wash the pipette with VS2 as explained in STEP2 (Fig. 3).

Perform ① and ② of STEP3 twice at diﬀerent positions in the VS2.

Conﬁrm the completion of VS equilibration by the below two criteria before loading oocytes/embryos on Cryotop.
・The oocytes/embryos are shrunk.
・The oocytes/embryos does not rise to the surface. (They stay in focus under microscope.)

If the above two points are not conﬁrmed, perform ① and ② again in no rush.

TIPSVS equilibration is performed eﬃciently by washing the pipette well with VS.

Referential operation time in VS1 and VS2 together is 60 - 90 seconds to ensure dehydration. It is recommended tokeep oocytes/embryos immersed in VS for at least 60 - 90 seconds.

Vitrification

7

Vitriﬁcation Protocol

PART5 Vitriﬁcation・Cryopreservation
Preparation

Prepare Cryotop with its black identiﬁcation mark facing up.

Fill up the container with liquid nitrogen as close to its maximum capacity as possible. This is preparation
to quickly plunge Cryotop into it.

1STEP1

3

Loading Oocytes/Embryos on Cryotop

Focus on the black tip mark of Cryotop (Fig. 1), expel the VS horizontally near the black mark, and place
the oocytes/embryoed (Fig. 2).

Fig.1

STEP2

2

Fig.2

Removal of Excess VS on Cryotop

Aspirate excess VS on the Cryotop from the extended drop edge, preferably from distal point from theoocytes or embryos (Fig. 3). The optimal liquid volume is such that the drop shadow is minimized ordisappeared(Fig. 4).

Fig.3Fig.4

TIPSPOINTWhen aspirating oocytes or embryos from the VS2, make sure to hold them at the tip of the pipette.

Make a droplet on the Cryotop by spreading the VS sideways. It prevents you from aspirating oocytes/
embryos while removing excel VS.

STEP3
8Vitrification

Conﬁrmation of Oocytes/Embryos

Remove the pipette from the Cryotop and conﬁrm if the oocytes/embryos are on the Cryotop.

STEP4

Rapid Cooling / Vitriﬁcation

STEP5

Capping Cryotop

Vitriﬁcation Protocol

Plunge the Cryotop into liquid nitrogen within 1 second for vitriﬁcation.

Keep the tip of the Cryotop in liquid nitrogen to cap it with the cover straw using forceps (Fig. 1).

Use your ﬁngers to hold the upper part of the cover straw and twist to cap it tight in air (Fig. 2 and 3).
Tip of the Cryotop must be kept submerged in liquid nitrogen .

保存Fig.1

STEP6

Fig.2

Fig.3

Make sure the Cryotop is securely capped. Use cane to store in a liquid nitrogen tank.

POINTTIP
Fill up the container with liquid nitrogen to enhance rapid temperature transition of Cryotop.

Information
Number of Oocytes/Embryos for one piece of Cryotop
Oocytes ：Maximum 4 per Cryotop.

Embryos：Depends on the number of embryos to be used after warming.

For example, if single embryo transfer is planned, store one embryo on one piece of Cryotop.

Vitrification

9Warming Protocol

Warming Procedure
Oocytes/Embryos

Preparation

Immersion in TS

Immersion in DS

Immersion in WS

Washing

PART1 Materials Required
1．Thawing Media VT602 / VT622 [Ref. 91121 / 91208]
No.1

TS (Thawing Solution): 4mL vial x 2

No.3

WS (Washing Solution): 4mL vial x 1

No.2

DS (Diluent Solution): 4mL vial x 1

2．35mm dish※Prewarm TS and 35mm dish to 37°C and equilibrate DS and WS to room temperature before use.

3．Repro Plate

4．Pipette for handling oocytes / embryos (hereinafter referred to as “pipette”)
5．Timer (with count up function)
6．Liquid nitrogen

7．Cooling Rack [Ref. 84014] for liquid nitrogen container
8．Forceps

9．Micro pipette: 300μL

10．Stereo microscope (Stage must be turned oﬀ).

CAUTIONFor optimal handling, please use a pipette with an appropriate internal diameter for oocytes (approximately140-180μm), or embryos (approximately 180-250μm).

All the warming procedures except for immersion in TS are performed at room temperature.

PART2 Preparation
STEP1

Warming TS

Prepare Thawing Media (VT602 / VT622).

Prewarm the TS vials capped and 35mm dishes to 37°C for the number of patients.

Required time: >30 min in an incubator and >5 min in a water bath (40℃). Do not warm more than 8 hours
to maintain the quality of the product.

10ThawingWarming Protocol
STEP2

Preparation of Cryotop

Remove the Cryotop from the storagetank and quickly transfer it to a container
ﬁlled with liquid nitrogen.

Remove the cover straw keeping theCryotop tip submerged in liquid nitrogen.

Place the Cryotop upright in the corner tokeep the closest proximity to the TS(Fig. 1 and 2).

Fig.1

TIP
Fill up the container with liquid nitrogen, so that Cryotop can be transferred into TS in a short distance forwarming.

Fig.2

STEP3

TS
entire volume

Preparation of the Dish

WS1300μL

DS300μL

WS2300μL

Prepare one DS well and two WS wells on Repro
Plate, respectively 300μL and cover with a lid.

After preparing the DS and WS, take out thewarmed TS vial and 35 mm dish from the incubator.
Pour the entire volume into the dish (Fig. 3).

POINTTIP

1

2

3

Fig.3

Invert the TS vial ﬁve times to mix its contents for its high concentration of cryoprotectants. The entire volume ofTS must be prepared in the dish.

STEP4

Setting of a Stereo Microscope

Focus on the center of the TS using the pipette inadvance (Fig. 4), and set the magniﬁcation of themicroscope to the minimum (widest ﬁeld of view).

Fig.4

Thawing11

Warming Protocol

PART3 Immersion in TS, DS and WS1
TSDSWS1

1 min（37℃）

3 min

5 min

Immersion in TS

STEP1Start the timer. (It is easier to check time with a count-up function).
Within 1 second, immerse the Cryotop sheet into TS.

Adjust the microscope focus on the tip of the sheet for oocytes/embryos. Continue observing even after
the oocytes/embryos naturally detach themselves from the sheet. Don’t touch it for one minute.

TIP
If the oocytes / embryos remain attached to the sheet after 1 minute in TS, gently expel some of the TS over them orgive vibration to the cryotop by ﬂicking its tip with a pipette, and let them detach. In either method, proceed withcare to avoid damaging the oocytes / embryos, regardless of the elapsed time in TS.

Immersion in DS

STEP2

After immersion in TS, aspirate the oocytes/embryos and additional TS for approximately 2 mm withthe pipette (Fig. 1).

Put the pipette tip in the center of the DS well bottom and place the oocytes/embryos in the TS layer
expelled in the DS. Leave it for 3 min (Fig.2).

DS2mm

TS

12Thawing

Fig.1

TSDS

3 min

Fig.2

Warming Protocol
STEP3

Immersion in WS1

Wash the pipette with DS.

Aspirate the oocytes/embryos and additional DS for approximately 2 mm with the pipette. Put thepipette tip in the WS1 well bottom and place the oocytes/embryos in the DS layer expelled in the WS1.
Leave it for 5min.

TIPS
Gradual osmolality change is important in the dilution process to ensure the previous solution mix
gently with a new one.

The “2mm” aspiration can be approximate. The purpose here is to bring in the previous solution tothe next. The width of Cryotop sheet is 1mm, hence a guide when making a 2mm guesstimate.

PART4 WashingSTEP1

Washing in WS2

Wash the pipette with WS2.

Aspirate the oocytes/embryos at the tip of the pipette (Fig. 1) and transfer them to the surface of WS2.
It is to minimize the volume of the WS1 carried into the WS2.

After the oocytes / embryos settle to the bottom, aspirate and transfer them to the surface of the WS2and wait for them to settle to the bottom (Fig. 2). No need to check the time here.

1Fig.1

STEP2

2

WS2

Fig.2

Recovery Culture

Perform recovery culture of oocytes/embryos after washing.
The estimated recovery culture time is as below:
Oocytes: 2 to 3 hours

Embryos: 30 min to 2 hours

The recovery culture for embryos is performed to conﬁrm the embryos's viability. Once survival is

conﬁrmed, proceed to the next procedure regardless of time. It takes 2 to 3 hours for oocytes spindles

to recover. Start ICSI or other following procedures after conﬁrming recovery of spindle or after 2 to 3
hours of recovery culture.

Thawing13

14

oocytes Vitrification Protocol

Oocytes Protocol

Vitriﬁcation
ES step

VS step

BS（20µL）＋ES（20µL）(First time)＋ES(20µL)(Second time)

ES（300µL）

Vitriﬁcation・Cryopreservation

VS1（300µL）
1VS2（300µL）

2

1

3Oocytes

2×53 min

3 min

DS step

WS step

TS（3.8mL）

DS（300µL）

WS1（300µL）

1 min 37℃

3 min

5 min

TS step

37℃×59 min

Room temperature

Warmingliquid nitrogen

60 - 90 sec

1WS2（300µL）

Room temperature2

Embryos Protocol

Vitriﬁcation
ES step

VS step

ES（300µL）

Vitriﬁcation・Cryopreservation

VS1（300µL）
1VS2（300µL）

2

1

3

liquid nitrogen

2×5×515 min

60 - 90 sec

Room temperature

Warmingembryos Vitrification Protocol

DS step

WS step

TS（3.8mL）

DS（300µL）

WS1（300µL）

1 min 37℃

3 min

5 min

TS step

1537℃

1WS2（300µL）

Room temperature

2Quality Results for Life

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