Cryodroppers for Mice 80010 & 80020 Resource Page


IMPORTANT! Before beginning the Cryodropper-E (embryo) 80010 and/or the Cryodropper-S (sperm) 80020 technique and procedure for mice, please read carefully:

Protocol for Cryodropper-E 80010 Vitrification of Mouse Embryos [PDF]
Protocol for Cryodropper-S 80020 Vitrification of Mouse Sperm [PDF]

Please watch the tutorial video for Cryodropper-E 80010 and Cryodropper-S 80020 technique and procedure for mice:

https://youtu.be/J3iSHdoJi2E

Cryodropper-E 80010 Instructions for Vitrification of Mouse Embryos

Vitrification of Mouse Embryos:

1. Embryos are kept in M2 at 37°C until cryopreservation.

2. Make vitrification and pre-vitrification media.

3. Set up portable LN₂ vapor phase freezer. [We use a Styrofoam cooler and float.]

4. Label each Cryodropper-E. [We color code or label just the bulb portion.]

5. Preload 100µl 0.5M Sucrose M2 in the Cryodropper-E bulb. Place the 100µl drop on a petri dish and pipette into the Cryodropper-E. Hold the Cryodropper-E by the open end and gently flick the media into the bulb portion.Gently squeeze the bulb to remove any media remaining in the embryo loading area and wipe the liquid off with a Kimwipe. Preload <5µl vitrification media into the center of the straw area by gently positioning a drop using the same method as before or with a gel loading tip. Store open end up in an Eppendorf rack.

6. On a cover of a 35mm dish, a drop of M2 (20-30μL per drop), a drop of pre-vitrification solution, and a drop of vitrification solution is loaded.

7. Label and precool the cryogenic storage vial. [We use a 4ml cryovial and label as needed. Qty 4 Cryodropper-E will fit per vial.]

8. Embryos are first transferred to the drop of M2 in the 35mm dish.

9. Prefill an embryo pipette with a small amount of the pre-vitrification solution. Under the dissecting microscope, transfer embryos from the M2 drop to the drop of pre-vitrification solution.

10. Incubate 30 seconds.

11. Prefill the embryo pipette with a small amount of vitrification solution. Transfer embryos from the previtrification solution drop to the vitrification solution drop.

12. Incubate 30 seconds.

13. Collect the embryos and load them into the vitrification solution preloaded in the Cryodropper-E. [It helps to focus the microscope on the embryos in the pipette.]

14. Seal Cryodropper-E at the end with a heat sealer. [Gently test to be sure the device is sealed, if not, seal again.]

15. Plunge Cryodropper-E into LN₂ until media in bulb freezes (@10 seconds). Store on raft in LN₂ vapor until all samples are processed. Transfer to vials.

16. The vial is then transferred to a LN₂ dewar and stored in the vapor phase.

Thawing of vitrified mouse embryos:

1. Prepare thaw dish for each Cryodropper-E as follows: a 60mm lid with space across the top (for 0.1 mL of 0.5 M sucrose in M2), and then 20-30µl drops of each clockwise: 0.5M sucrose in M2, 0.25M sucrose in M2, and M2 (2 drops). Prepare incubation dish as follows: 60mm dish with 100µl KSOM elongated drop through center, 30µl KSOM drop on either side for washing embryos prior to culture. Cover with paraffin and equilibrate at 37°C with 5% CO₂ for at least 30 min.

2. The Cryodropper-E containing vial is removed from the storage vessel and kept in LN₂ vapor until ready for thaw.

3. The Cryodropper-E is immersed in a water bath at 37˚C until the media in the dropper is thawed (@10 sec).

4. The tip of the Cryodropper-E is cut off and the embryos are deposited onto the culture dish. The 0.5M sucrose M2in the Cryodropper-E bulb is gently flicked down to the straw and expelled from the Cryodropper-E into the embryo drop, minimizing bubbles. The embryos are immediately collected from the drop using an embryo pipette prefilled with 0.5M sucrose in M2.

5. The embryos are transferred to a new drop of 0.5M sucrose in M2 followed by an incubation of 2 min.

6. Using an embryo pipette prefilled with 0.25M sucrose in M2, the embryos are transferred quickly to the drop of 0.25M sucrose in M2 and are incubated for another 2 min.

7. Using an embryo pipette prefilled with M2, the embryos are transferred quickly to the first drop of M2 and are incubated for 1 min.

8. The embryos are then washed through the last drop of M2, through the 2 drops of KSOM, and are then transferred to the long drop of KSOM under oil.

9. Embryos are cultured at 37°C with 5% CO₂ (for 2 days for late morula or blastocyst stage).

References: Adapted by B. Stone from Wai Hung Tsang and King L. Chow, BioTechniques Protocol Guide 2010 (p. 55) doi10.2144/000113258.

 

Cryodropper-E 80010 and Cryodropper-S 80020 Video: https://youtu.be/J3iSHdoJi2E

 

Equipment:

• Cryodropper-E for Embryo Vitrification (black line: ParaTechs 80010)
• Cryovial (4 ml) or other suitable LN₂ storage option (USA Scientific 1440-9100)
• CO₂ incubator at 37˚C
• Slide warmer or 37˚C incubator
• Water bath @ 37˚C (500ml beaker of water in a 37˚C incubator works well)
• Pipettors and tips; ex: 1ml, 200ul, 20ul, 2ul
• Eppendorf tubes, racks
• Portable LN₂ vapor phase freezer with Styrofoam raft (see photo below)

• Fine tip Sharpie for labeling
• Kimwipes
• Tissue culture dishes; 35mm, 60mm
• Embryo handling pipettes and mouth pipettor
• Stereomicroscope with adjustable light source
• Timer
• Impulse heat sealer (American International Electric AIE-105T)
• Forceps
• Scissors
• LN₂ vapor phase storage dewar
• 0.22µm filter units for sterilization (ex: Millipore SCGVUORE and SLGP033RS)
• Refrigerator/freezer for media storage

 

Media and Reagents Required:

M2 medium (Millipore MR-015-D)
Ficol PM70 (Sigma-Aldrich F2878)
Sucrose (Sigma-Aldrich S1888)
Ethylene Glycol (Sigma-Aldrich 102466)
Dimethyl sulfoxide (DMSO)(Sigma-Aldrich D2650)
• 0.5 M sucrose
• FS
• Pre-VS
• VS
• 0.25M sucrose
Paraffin Oil (Sigma-Aldrich 18512)
KSOMᴬᴬ medium (Millipore MR-121-D)

 

Cryodropper-E 80010 Recipes: 


PARATECHS CORPORATION LIMITED WARRANTY

ParaTechs warrants that, at the time of shipment, the Product will conform to the specifications that accompany the Product. This warranty limits ParaTechs’ liability to replacement of the Product.  PARATECHS MAKES NO OTHER WARRANTIES, EXPRESSED OR IMPLIED, WITH RESPECT TO THE PRODUCT; INCLUDING ANY WARRANTIES OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE OR THAT THE PRODUCT DOES NOT INFRINGE ANY PROPRIETARY RIGHTS OF ANY THIRD PARTY.

 

Cryodropper-S 80020 Instructions for Cryopreservation of Mouse Sperm

Sperm Freezing:

1. Prepare 2 male mice (>8 weeks old with proven fertility) by mating (plug positive) 4-7 days prior to sperm cryopreservation.

2. Prepare 1 sperm dish per mouse by depositing a 60μl drop of CARD CPA on a 35mm dish. Cover the drop with paraffin oil. Add a second 60μl aliquot of CPA to the first drop to make a tall, semi-spherical drop of CPA. Equilibrate at 37 ̊C (not in CO₂).

3. Label each Cryodropper. [We color code or label just the bulb portion.]

4. Prepare Cryodroppers: prepare 90μl drops of CARD PM medium in a dish, 1 drop per Cryodropper. Let equilibrate at 37 ̊C and 5% CO₂ for 10 min. Load drops into droppers by pipetting and gently flick the medium to the bulb portion. Gently squeeze the bulb to remove any medium remaining in the sperm loading area and wipe the liquid off with a Kimwipe. Store open end up in an Eppendorf rack at 37 ̊C with 5% CO₂ until sample loading.

5. Set up the freezing box with LN₂ and allow to cool.

6. Label and precool the cryogenic storage vials. [We use 4ml cryovials and label as needed. 4 Cryodroppers will fit per vial.]

7. Euthanize the mice.

8. Remove the cauda epididymides. Place them on a Kimwipe and, under a microscope, completely remove all fat and blood.

9. Transfer one epididymis from each male to each sperm dish. This keeps the sperm from two males mixed in the sperm dishes. Note: the following steps must be performed quickly; under 30 minutes total from sperm release to freezing.

10. Using watchmaker’s forceps and small angles scissors, make at least 6 incisions in each epididymis.

11. Place dish on slide warmer at 37 ̊C for 3 minutes. Rotate dish every minute to disperse sperm from tissue. Gently squeeze remaining sperm from the tissue as the tissue is removed from the medium.

12. Loading Cryodroppers: Using a gel loading pipette, carefully load the straw portion with 10μl sperm suspension in the center. Seal with a heat sealer. Put loaded prototypes directly on float in LN₂ vapor for 10 minutes.

13. Transfer Cryodroppers to vials. The vials are then transferred to a LN₂ dewar and stored in the vapor phase.

 

Sperm Thaw:

1. Remove Cryodropper from LN₂ storage.

2. Immerse in a 37 ̊C water bath until thawed and incubate for 10 minutes.

3. Remove device from water bath and gently dry with a Kimwipe.

4. Using scissors, cut the tip of the Cryodropper off and transfer sperm drop to a fresh IVF dish.

5. Gently shake CARD PM medium to end of device with single flick of wrist. [Too much force and the medium will be lost.]

6. Apply medium to sperm drop. [Note: If an IVF dish is used, the humidity should be maintained by water in the outer chamber. If a regular petri dish is used, the sperm drop should be covered with Paraffin equilibrated in the CO₂ incubator for at least 30 minutes prior to sperm thaw.]

7. Incubate in the CO₂ incubator at 37 ̊C to capacitate. [We generally capacitate ~45 minutes.]

 

References:

Adapted by B. Stone from Behringer R, Gertsenstein M, Vintersten K, Nagy A. 2014.Manipulating the mouse embryo: a laboratory manual, 4thed. Cold Spring Harbor (NY): ColdSpring Harbor Laboratory Press.

 

Cryodropper-E 80010 and Cryodropper-S 80020 Video: https://youtu.be/J3iSHdoJi2E

 

Media Required:

• Paraffin Oil, (Sigma-Aldrich 18512)
• FERTIUP cryoprotectant (CPA) (Cosmobio KYD-001) (Can be made in-house as well, Behringer et al., pg. 674-675)
• FERTIUP preincubation medium (PM) (Cosmobio KYD-002) (Can be made in-house as well, Behringer et al., pg. 614)

 

Equipment:

• Cryodropper for Sperm Vitrification (red line: ParaTechs 80020)
• Cryovial (4 ml) or other suitable LN2 storage option (USA Scientific 1440-9100)
• CO₂ incubator at 37 ̊C
• Slide warmer or 37 ̊C incubator
• Water bath @ 37 ̊C (500ml beaker of water in a 37 ̊C incubator works well)
• Gel loading tips (ex: USA Scientific 1022-0600)
• Pipettors and tips; ex: 1ml, 200ul, 20ul, 2ul
• Eppendorf rack
• Portable LN₂ vapor phase freezer with Styrofoam raft (see photo below)

• Sharpie for labeling
• Kimwipes
• Tissue culture dishes; 35mm, IVF dishes (optional)
• Stereomicroscope
• Timer
• Impulse heat sealer (American International Electric AIE-105T)
• Forceps (Watchmakers #5)
• Scissors, dissection and small angled
• LN₂ vapor phase storage dewar

 

Animals:

• Male mice for sperm collection (proven fertility, mated 4-7 days prior to collection)

 

PARATECHS CORPORATION LIMITED WARRANTY

ParaTechs warrants that, at the time of shipment, the Product will conform to the specifications that accompany the Product. This warranty limits ParaTechs’ liability to replacement of the Product.  PARATECHS MAKES NO OTHER WARRANTIES, EXPRESSED OR IMPLIED, WITH RESPECT TO THE PRODUCT; INCLUDING ANY WARRANTIES OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE OR THAT THE PRODUCT DOES NOT INFRINGE ANY PROPRIETARY RIGHTS OF ANY THIRD PARTY.

 

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