Ovarian Tissue
Vitrification Protocol

Products -Ova Cryo Kit
QR codeVitriﬁcation protocol

Ovarian tissue cryopreservation ﬂowCooling(Vitriﬁcation)

Equilibration of Vitriﬁcation Solution

Ovarian Tissue Processing

Cryopreservation

PART1 Materials Required
1．Ova Cryo Kit (Code. VT301S)
Cryo1：20 mL×1
Cryo2：20 mL×1
Cryo3：20 mL×1

＊1
＊2
＊3

For the processing of ovarian tissue (Method 2)
For Open System
For Closed System

2．Ova Rinse (Code. OVR-100)

3．Square Measure (Code: Square Measure)＊1

4．Ova Cryo Device Type M (Code：ODT×10)＊25．Ova Cryo Closed Device CryoSheet (Code：OCS×10)＊3
6．CryoSheet Cane (Code：CryoSheet Cane×10)＊3
7．1~10mL syringe ×1
8．18~21G needle ×1

9．Spitz or centrifuge tube for follicular ﬂuid collection

10．Dish for oocyte inspection

11．Microscope for oocyte inspection

12．Dish (OD 60mm) ×3 : for Cryo1, 2, 3

13．Dish (OD 60mm - 100mm) ×2 : for immersing the ovary in Ova Rinse

14．Scissors Note : 2 types of scissors ; straight-end and curved-end facilitate the preparation of ovarian tissue.
15．Curved tweezers (about 12cm)
16．Tweezers (about 20cm) ×2

17．Surgical knife (Blade No. 10)
18．Sterilized gauze

19．Count-up Timer
20．Liquid nitrogen

21．Liquid nitrogen container (Code: Cooling Rack)
22．Microtome blade with handle＊1
23．Microtome blade＊1

24．Surgical knife (Blade No.11)＊1
25．Cane (C-2 Cane)＊2

26．Sealer＊3 Note : Tabletop and temperature adjustable sealer, capable of sealing dimensions over 5cm.
For example, Fujiimpulse P-200.

2

Vitrification

Vitriﬁcation protocol

Ova Cryo Kit（Ref. 82212

Ova Rinse（Ref. 82215

Code：VT301S）

Code：OVR-100）

Ova Cryo Device Type M（Ref. 81213

Code：ODT×10）

Cryo1

Cryo2

Cryo3

Square Measure（Ref. 81212

Code：Square Measure）

Ova Cryo Closed Device CryoSheet（Ref. 81214

Code：OCS×10）

Ova CryoSheet Cane

（Ref. 81215

Code：KOCC10）

Vitrification

3

Vitriﬁcation protocol

PART2 Preparation
Preparation

Allow Cryo1, Cryo2, and Cryo3 to reach room temperature (25 to 27℃).

Label the dishes with "Cryo1," "Cryo2," and "Cryo3" using a labo marker.

Pour the entire contents of each corresponding liquid into the labeled dish.

STEP1Wash the extracted ovarian tissue with Ova Rinse to remove excess blood.

STEP2

Pierce the follicles with an injection needle and aspirate theﬂuid with a syringe (Figure 1).

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

follicular ﬂuid
1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

TIP
The immature oocytes retrieved are vitriﬁed after in vitro maturation (IVM). Mature oocytes are either vitriﬁed
for preservation or used for in vitro fertilization (IVF).

4Vitrification

0.2

0.1

Fig.1

Vitriﬁcation protocolPART3 Dissection of Ovarian Tissue
Method 1

Carefully proceed with the following steps using Ova Rinse, ensuring the ovarian tissue does not dry out.

STEP1

Using straight scissors, cut the ovaries in half along theequatorial plane (Figure 1).

ovarian equatorial plane

CUT

STEP2

Fig.1

Divide the ovary in half, open it with the medullary side facing
upward to prevent drying, immerse it in Ova Rinse, and startﬁne sectioning (Figure 2).

Fig.2

STEP3

Using curved scissors, remove the medulla from the opened ovary, leaving
only the cortex. Gently hold the ovary with tweezers, and remove themedulla with curved scissors, repeating until the cortex is 1mm thick(Figure 3). Inserting the curved scissors in a scooping motion facilitateseasy removal of the tissue.

Fig.3

STEP4

Once you have cortex without medulla, use a surgical knife andcut it into 1cm x 1cm size (Figure 4). Removing the medulla after
slicing makes the process easier.

1cm square with a thickness of 1mm

Vitrification

Fig.4

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Vitriﬁcation protocol

Method 2

Carefully proceed with the following steps using Ova Rinse, ensuring the ovarian tissue does not dry out.
If processing the ovarian cortex using Method 1 is challenging, please use the Square Measure.
The Square Measure comprises parts A and B (Figure 1).

Square Measure A

Preparation
STEP1

Square Measure B

Fig.1

Wipe oﬀ any excess wetness from the ovarian surface with sterile gauze to prevent theSquare Measure from slipping.

Place Square Measure A on the area of ovarian surface where you plan to slice.

With a blade (blade No. 11), create incisions in a pattern along the inner frame ofSquare Measure A, ensuring the cuts intersect at the corners and penetrate to a depth of at least 1 mm.

TIP
Square Measure Aovaryovary

Square Measure A

Square Measure A

Be sure to cut ovary from inner edge ofSquare Measure A.
(cut by drawing Sharp “#” line)

STEP2

Attach Square Measure B to Square Measure A and place it on the ovary surface (Figure 2).

Square Measure B

Metal stopper

Square Measure A
Fig.2

Ovary

STEP3
6

Vitrification

Side view

Microtome blade

Fig.3

Chipped corner

While holding down Square Measure from above, insert themicrotome blade between Square Measure A and B, andsmoothly slice the tissue until it reaches the metal stopper (Figure 3).

Square Measure B

Microtome blade

Square Measure AovaryTop view

Vitriﬁcation protocol

PART4 Equilibration
Cryo1

5 min

Cryo2

Gauze

5 min

Cryo3

Gauze

15 minMultiple ovarian sections can be processed simultaneously with Cryo1, Cryo2, and Cryo3.

STEP1

Wipe oﬀ excess wetness from the ovarian sections with gauze and immerse them in Cryo1 for 5 minutes.

STEP2

After equilibrating in Cryo1, gently wipe oﬀ excess Cryo1 solution from the ovarian sections with gauze,

STEP3

After equilibrating in Cryo2, gently wipe oﬀ excess Cryo2 solution from the ovarian sections with gauze,then transfer them to Cryo2 and allow them to equilibrate for 5 minutes.

then transfer them to Cryo3 and allow them to equilibrate for 15 minutes.

TIP
Prepare the Device (ODT or OCS) and liquid nitrogen while the ovarian tissue is equilibrating.

Vitrification

7

Vitriﬁcation protocol

PART5 Vitriﬁcation for Cryopreservation

Open System

Preparation
STEP1

Prepare the same number of Ova Cryo Devices (ODTs) as the number of prepared ovarian tissues.
Ensure all ODT vials are labeled with patient IDs and necessary information.

Wipe the ovarian sections with gauze. Spread them out to maximize their surface
area and place them on the metal part of the device. Position the ovarian

sections near the tip of the metal part, avoiding the base where the metal is

5mm

Fig.1

embedded in the cap, and leave approximately 5 mm of space to place theovaries (Figure 1).

Liquid nitrogen

STEP2

Using tweezers, hold the device cap and quickly immerse it in liquid

STEP3

After the ovarian sections have cooled, attach the vial to the cap and seal it while

STEP4

Place the device into a cane and store it in the liquid nitrogen tank (Figure 4).

nitrogen (Figure 2).

immersed in liquid nitrogen (Figure 3).

Fig.2

Fig.3

Fig.4

POINT
TIP
Immerse the device into liquid nitrogen with the ovarian sections placed facing downward to prevent themfrom detaching.

If ovarian sections detach from the device in liquid nitrogen, place the sections directly into the vial and cap it.

8Vitrification

Closed SystemPreparation

Vitriﬁcation protocol
No:
ID:
Day:Label the patient information on the Ova Cryo Sheets for the number ofovarian sections, as well as on the corresponding Ova Cryo Sheet pouches(Figure 1).

Fig.1

STEP1

Unroll the ﬁlm portion. Use gauze to wipe the ovarian sections andplace them within the frame of the device to maximize their surface
area (Figure 2).

Fig.2

STEP2

Place the ﬁlm over the ovarian sections, ensuring they are fully covered,and carefully seal it to remove any excess air.

Ova Cryo SheetSTEP3

Place the device into the pouch designed for Ova Cryo Sheets.

Remove air, lightly press the pouch with your ﬁngers, and seal it using
a heat sealer along the black line (Figure 3). Please ensure the pouchis sealed at the correct temperature, as excessive heat from the sealer

SEAL

No:
ID:
Day:

Ova Cryo Sheetpouches

could potentially damage it.

It is recommended that the temperature is checked in advance with aFig.3

spare pouch.

STEP4

Using tweezers, swiftly immerse the device in liquid nitrogen.

After immersing, gently move around the device in liquid nitrogen toNo:
ID:
Day:

Liquid nitrogenensure rapid cooling (Figure 4).

Fig.4

STEP5

Ova Cryo Sheet Cane

Place the device into a cane and store it in the liquid nitrogen tank(Figure 5).

Fig.5

Vitrification

9Warming Protocol
Ovarian Tissue Warming ﬂow
Ovarian Tissue Section Thawing

Dilution and WashingTransplantation/Culturing

PART1 Materials Required
1．Ova Thawing Kit (Code. VT302S)＊1

Thaw1：100mL ×1

For Closed System

Thaw2：20mL ×1
Thaw3：20mL ×1

2．Ova Culture (Code. OVCL-100)

Or Ova Culture with HEPES (Code. OVCM-100) for additional culture

3．Liquid nitrogen

4．Liquid nitrogen with its container (Code. Cooling Rack)
5．Tweezers (about 20cm)：for thawing procedure
6．Water bath

7．Dish (OD 60mm - 100mm) ×2
8．Container (110mL / 4.5 oz.)

9．Tweezers (about 12cm)：for dilution and washing of ovarian tissue

10．Count-up Timer

11．Scissors＊1：for cutting the pouch

Ova Culture（Ref.82216

Ova Thawing Kit（Ref.82222

10Thawing

Code：VT302S）

Thaw1

Thaw2

Thaw3

Code：OVCL-100）

Ova Culture with HEPES（Ref.82217

Code：OVCM-100）

Warming Protocol

PART2 Preparation
STEP1

Warm Thaw1 bottle to 37℃ in water bath and pour the entire volume of Thaw1 into a sterilized container.
Allow Thaw2 and Thaw3 to reach to room temperature (25-27℃).

Label each container or dish with "Thaw1," "Thaw2," and "Thaw3" using a labo marker, and pour thecorresponding liquid into each dish.

TIP
To facilitate rapid transfer of the freezing device from liquid nitrogen to Thaw1 during warming, place theliquid nitrogen container right next to the water bath.

Thawing11

Warming Protocol

PART3 Warming
Open System
Thaw1

1 min

Thaw2

Thaw3

3 min

5 min

STEP1

Remove the device from the tank and transfer it to a liquid nitrogen container. Using tweezers, carefully

STEP2

Quickly transfer the device to Thaw1 and gently move around within Thaw1 then equilibrate for 1

remove the cap of the device in the liquid nitrogen. Conﬁrm the presence of ovarian sections.

minute. Remove the device promptly upon detachment of ovarian sections.

TIP
To maintain the temperature of Thaw1, immerse only the metal part of the device in it.

STEP3

After equilibrating in Thaw1, transfer the ovarian sections to Thaw2 and immerse them for 3 minutes.

STEP4

After equilibrating in Thaw2, transfer the ovarian sections to Thaw3 and immerse them for 5 minutes.
Once warming is completed with Thaw3, proceed to the next procedures such as transplantation.

Following warming, perform an additional 30-minute culture using Ova Culture with HEPES before

proceeding with transplantation. If additional culture exceeding 30 minutes is necessary, utilize OvaCulture and conduct it in the incubator.

12ThawingWarming Protocol

Closed System
Thaw1

1 min

STEP1

Thaw2

Thaw3

3 min

5 min

Take out the frozen device from the storage tank and put it into theliquid nitrogen container. While immersed in liquid nitrogen, cut along

CUT

No:
ID:
Day:

the red line of the pouch and open it.

Make sure the top of the pouch is positioned above the liquid nitrogenlevel to avoid any liquid nitrogen from getting inside (Figure 1).

Fig.1

STEP2

Take the frozen device out of the pouch and quickly move it to Thaw1, allowing it toSTEP3

After equilibration with Thaw1, transfer the ovarian sections to Thaw2 and immerse for 3 minutes.

STEP4

After equilibration with Thaw2, transfer the ovarian sections to Thaw3 and immerse for 5 minutes.

equilibrate for 1 minute. To maintain the temperature of Thaw1, immerse only the ovarian section ofthe frozen device in it. Once the ovarian section is detached, take out the device from Thaw1.

Once warming is completed by Thaw3, proceed to the next procedures such as transplantation.

Following warming, perform an additional 30-minute culture using Ova Culture with HEPES before

proceeding with transplantation. If additional culture exceeding 30 minutes is necessary, utilize Ova
Culture and conduct it within the incubator.

Thawing13

Ovarian Tissue Protocol
Vitriﬁcation

Cryo1Warming

5 minGauze

Cryo2

Ova Cryo Device Type M

Ova Cryo Sheet（Open System）

5 min（Closed System）
CUTGauze

Cryo3

Liquidnitrogen

15 minLiquidnitrogen

Gauze

Ova Cryo Device Type M（Open System）

Ova Cryo Sheet（Closed System）

Thaw1（37℃）

Thaw1（37℃）

1 min
SEAL

1 min

No:
ID:
Day:

Thaw2
No:
ID:
Day:

Liquidnitrogen

Liquidnitrogen

3 min

Thaw3
5 min

14

Ovarian Tissue Protocol

No:
ID:
Day:

Ovarian Tissue Protocol

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Quality Results for Life

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