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A simple and efficient vitrification procedure for

cryopreservation of mouse embryo and oocyte in straw Based on a method originally published by Nakagata et al (1997)

1. Materials

1.1. Media and solutions

1M DMSO solution (in PB1 medium)

DAP213 vitrification solution (in PB1 medium)

0.25M Sucrose (in PB1 medium)

KSOM+AA for embryo recovery from vitrification and in vitro culture

1.2. Equipment

60mM Culture Dishes (Cat. No. PAA10060X, PAA Laboratories Ltd)

35mM Culture Dishes (Cat No. 351008; Falcon)

Vitrification Cooling Plate (VCP, Elim Springs Biotech Ltd.)

Plastic Sandwich Box – Base & Lid (Part No. 6107008; Azpack Ltd.)

0.25mL French Straws (Cat No. FZA201; Planer PLC.)

20µL, 200µL and 1.0mL Pipettes and Tips (i.e. Gilson)

10µL wedged pipette tips (Axygen Inc; T-400)

1.0mL/10mL Syringes

0.2µm Syringe filters (Cat No. 514-7011,VWR)

Mouth Pipette

Microscope

Stopwatch

Liquid Nitrogen (flask)

Straw Rods

Labels for straws

Heat Sealer

Perspex Support (for straws)

2. Preparation for vitrification

2.1. Fill the sandwich box with crushed ice and place the Vitrification Cooling Plate on top. The

temperature of VCP will reach to 0°C in 10 seconds.

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2.2. Preparation of straws

Push the cotton/polyvinyl alcohol plug into straw, from position A to position B,

using a metal rod with a stop.

Place the straws on a Perspex support. Using a permanent marker pen, make two

calibration marks using the guidelines on the plate to obtain the correct distances.

The calibration marks should be placed at 11mm and 25mm intervals from the end

of straw (opposite the PVA plug end). Then label the straws and place them on VCP.

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2.3. Preparation of dishes

Label three 60mM culture dishes (DMSO, DAP213, and Sucrose) and place the DAP213 and

Sucrose dish onto the VCP. Then Pipette 45µL drops of DAP213 and 0.25M Sucrose into the

corresponding dishes, the number of drops will be the same as the number of straws.

2.4. Filter the 1M DMSO and place 80µl drops of 1M DMSO into the base of 60mm

culture dish. One is to wash the embryos, while the others are to hold the embryos.

3. First step: pre-treat embryos in 1M DMSO solution

Load the mouth pipette with 1M DMSO solution and transfer the embryos from the

culture medium into one of the drops of DMSO. The embryos float in the drop and

then sank to the bottom in 1-2mins. Once the embryos have settled, split them

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between the other drops. Once this is done, place the DMSO dish on the VCP for

5min.

4. Second Step: embryos exposed to the DAP213 solution

After 5min has elapsed, move the DMSO dish to a microscope and use a wedge-

shaped 10μl pipette tip (Axygen Inc; T-400) to transfer 5μl of 1M DMSO containing

the embryos to a 45μl of DAP213 drop. The embryos were placed on the VCP for

another 5min. (Tip: to stop the embryos sticking to the inside of the pipette, pick up 1µL of

DMSO first then the embryos).

After 1min in the DAP213 drop, start to load the embryo into the pre-cooled straws.

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Attach a 1ml syringe to the labelled end of the straw and aspirate 0.25M Sucrose

solution, until the meniscus reached mark 1. Then aspirate air, to move the sucrose

meniscus to mark 2. Then aspirate entire drop of DAP213 containing embryos into

the straw. Aspirate air until the sucrose meniscus has been drawn half way along

the cotton/PVA plug.

Place the straws back on VCP for at least 30 seconds before the unlabelled end of

the straws is sealed. Once the 5min equilibration time has elapsed, plunge the

straws into LN2, and transfer the straws to a secure LN2 location.

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Note: Equilibrating embryos in DAP213 should be conducted at 0°C. This is the most

important factor in the procedure. Therefore, if you want to get the best and stable

recovery from this method, the straws and DAP213 dish should be kept on the VCP as long

as possible or exposed to room temperature as short as possible in the procedure.

5. Warming the vitrified embryos

5.1. Place a tube of 0.25M Sucrose warming solution (1ml) in an incubator (37°C) for at

least 5min before warming;

5.2. Add 0.5ml 0.25M sucrose (pre-warmed to 37°C) into a 60mm culture dish, and place

the dish on a 37°C warmed stage;

5.3. Remove the required straw from the storage tanks and place in a flask of LN2.

5.4. Using forceps take the straw from LN2 and place it in a 37°C water bath. Once the

DAP213 fraction has melted remove the straw immediately from water bath

(Usually it takes 3-5 seconds).

5.5. Wipe the straw with a tissue, then holding the straw firmly and horizontally, cut

through middle of the cotton/PVA plug.

5.6. Cut off the heat sealed end of the straw, then holding the straw vertically over the

drop of 0.5ml 0.25M sucrose, use a metal rod to push down the remaining

cotton/PVA plug, expelling the contents of the straw into the drop. Do not allow the

tip of straw to touch the drop.

5.7. Gently rotate the dish to mix the solution.

5.8. Set a countdown timer for 3mins. Collect the embryos from the solution, and

transfer them into a drop of KSOM+AA, then keep them in an incubator (37°C, 5%CO2

in air) for 10mins.

5.9. Wash the embryos through another two KSOM+AA drops before the embryos are

assessed for damage.

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5.10. After the final wash the embryos are ready to be transferred into the recipient

female. Follow the surgical embryo transfer procedures described by Nagy et al.

5.11. If you are not ready to set up the embryos transfers the embryos should be kept in

KSOM+AA drop and held in the incubator until required.

6. Reagents and solutions

Composition of PB1 medium. Osmolality should be 285-295mOsm.

Reagent Name mg/100ml Vendor Catalogue Number

NaCl 800 Sigma S-5886

KCl 20 Sigma P-5405

CaCl2 12 Sigma C-5670

KH2PO4 20 Sigma P-5655

MgCl2 6H2O 10 Sigma M-2393

Na2HPO4 115 Sigma S 5136

Na-Pyruvate 3.6 Sigma P-4562

Glucose 100 Sigma G-6152

Penicillin 7.5 Sigma P-4687

Streptomycin 5.0 Sigma S-1277

BSA 300 Sigma A-4378

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Tips for home making PB1 medium from PBS:

(1) PBS (-)

Dissolve 9.6 g Dulbecco's PBS (-) in double distilled water (culture grade water) with a final volume of 980 ml.

Sterilize the solution by autoclave (121oC, 15 min.) or filtration (22 µm filter).

Store in the refrigerator at 4 oC.

(2) cation solutions

a) CaCl2·2H2O (100x concentration)

Dissolve 132 mg CaCl2·2H2O in 10 ml of double distilled water.

Sterilize the solution by filtration.

b) MgCl2·6H2O (100x concentration)

Dissolve 100 mg MgCl2·6H2O in 10 ml of double distilled water.

Sterilize the solution by filtration.

(3) PBS (+)

Add 10 ml of CaCl2·2H2O solution to 980 ml of PBS (-) solution.

Mix gently.

Add 10 ml of MgCl2·6H2O solution and mix gently.

Store in the refrigerator.

(4) PB1 (BSA free)

Add the following chemicals to 100ml PBS (+) solution described above.

Na Pyruvate 3.6 mg Sigma (#P-8574)

Penicillin G 6.4 mg

Sigma (#P-4687)

Glucose

100 mg

Wako (#041-00595)

(5) PB1 medium

Add 300 mg of BSA (Bovine Serum Albumin) to 100ml of PB1 (BSA free),

and let it sit until dissolved.

Sterilize the solution by filtration and store in the refrigerator.

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1M DMSO solution:

Prepare the 1M DMSO solution by adding 1.56ml DMSO into 18.44ml of PB1 medium. Filter

the solution through a 0.22µm syringe end filter. This solution has a shelf life up to 3 month

at 4°C.

0.25M Sucrose for warming:

Prepare the 0.25M sucrose solution by adding 1.711g of sucrose to 20ml of PB1 medium.

Filter the solution through a 0.22µm syringe end filter. This solution has a shelf life up to 3

month at 4°C.

DAP213 vitrification solution:

Composition of DAP213 vitrification solution

Solution A

Reagent Name ml/10ml Vendor Catalogue Number

PB1 2.31 - -

DMSO 3.13 Sigma D-2650

Propylene glycol 4.56 Sigma P-1009

Solution B

Reagent Name mg/10ml in PB1 Vendor Catalogue Number

Acetamide 1181.4 Sigma A-0500

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Tips for Home making DAP213 solution:

DAP213 Preparation

Solution A (25ml) Solution B (25ml)

15ml PB1

2.95g Acetamide

Make up to 25ml

5ml PB1

4M DMSO (7.10ml)

6M PG (10.975ml)

Make up to 25ml

Mix by gently stirring

Mix by gently stirring

Mix by gently stirring

Finished solution of DAP213 (50ml)(Mix by gently stirring)

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Filter the solution through a 0.22µm syringe end filter. This solution has a shelf life up to 3

month at 4°C.

This media can be purchased commercially from Cosmo Bio Co., Ltd (www.cosmobio.com).

7. Flowchart for DAP213 vitrification

Embryos in 1M DMSO in dish at room temperature

Embryos in 1M DMSO in dish at 0⁰C

Embryos in DAP213 in dish at 0⁰ C

Plunging into LN2

The procedure of Vitrification

Take the straw from LN2 and place it in a 37⁰C water bath

Embryos in DAP213 in straw at 0⁰ C

2-5min

5min (<20min)

After1min load the straws

5min (<10min)

3-5 second

2-5min

10min

Expel the contents of straw into 0.25M Sucrose drop

Embryos in KSOM or washing & recovery

Embryos viability is assessed and is ready for use

The procedure of Warming

8. Reference

Nakao, K., Nakagata, N., and Katsuki, M. 1997. Simple and efficient vitrification

procedure for cryopreservation of mouse embryos. Experimental animals / Japanese

Association for Laboratory Animal Science 46:231-234.

Nagy A., G.M., Vintersten K., Behringer R. 2003. Manipulating the Mouse Embryo - A

laboratory manual (3th Edition). Cold Spring Harbor Laboratory press, New York.