Reviewed 6/23/2004

Supplies for thaw

• Liquid nitrogen Dewar
• 25 degrees Celsius water bath
• Scissor
• Kim Wipe
• Metal plunger (enclosed)
• Timer
• Forceps
• Pipetter
• 10 - 35mm Falcon dishes containing 1 ml of M2
• M2 - See enclosed media sheet

Thaw protocol

The procedure used for freezing and thawing in The Jackson Laboratory Frozen Embryo Repository is based on that described by Renard, J.P. and Babinet, C. (1984). High survival of mouse embryos after rapid freezing and thawing inside plastic straw with 1-2 propanediol as cryoprotectant. Journal of Experimental Zoology 230, 443-448

Your embryos should be transferred into your clean unit as soon as possible.

Reviewed 6/23/2004

Supplies for thaw

• Dewar containing the sample to thaw
• Vial rack
• 1ml pipetter
• Forceps
• 10 - 35mm Falcon dishes containing 1 ml of DPBS
• Media

Thaw protocol

The procedure used for freezing and thawing in The Jackson Laboratory Frozen Embryo Repository is based on that described by Whittingham, et al. [Whittingham, D., S. Leibo, et al. (1972). Survival of mouse embryos frozen to -196°C and -269°C. Science. 178:411-14.]

Embryos require a slow thaw:

1. To thaw, remove vials from liquid nitrogen and place in a rack at room temperature until completely thawed (this will take about 15-20 minutes).
2. SLOWLY pipette 800ul of DPBS, drop wise into each vial to dilute the DMSO.
3. Using a 1ml pipetter set at 1ml, suck up about half the solution and then expel slightly (to dislodge any embryos that may be stuck to the side of the vial) and then continue removing all the solution and placing it into a small petri dish.
4. Wash the embryos through 10 dishes of DPBS, using a new pipette for each wash (each dish should contain 1ml of media). 
5. Your embryos should be transferred into your clean unit as soon as possible.

Phosphate buffered saline (modified Dulbecco's)

1. For a final volume of 100 ml of solution weigh out the following into approximately 75 ml of culture grade water:

2. Dissolve 10.0 mg CaCl₂ (anhydrous) in about 10 to 20 ml of culture grade water.
3. Add the dissolved CaCl₂ to the above ingredients and bring the total volume up to 100 ml.
4. Use 20 ml of the above solution for making up a 2 M solution of DMSO (Aldrich).
5. Add 240.0 mg of crystallized Bovine Serum Albumin (BSA) and 0.1 ml of a 1% solution of Phenol red to the remaining 80 ml.
6. Sterilize the solutions by filtration.

HTF

Add each chemical in order and mix carefully.

Gas for 5 minutes with 5% CO2, 5% O2 balanced with N2, using a 1 ml pipette submerged in the media before adding BSA. Add BSA after gassing and then incubate to dissolve. Filter with .22um filter and gas before refrigerating.

Always gas the medium after every use.

Reference: Quinn P, Kerin JF and Warnes GM (1985) Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertility and Sterility 44: 493-498.

KSOM medium

1. For a final volume of 100 ml of medium weigh out the following into approximately 75 ml of double distilled water:

2. Dissolve 25 mg CaCl₂ (anhydrous) in about 25 ml of double distilled water.
3. Add the dissolved CaCl₂ to the above ingredients and bring the total volume up to 100 ml.
4. Add 0.1 ml of a 1% solution of Phenol red to the solution.
5. Gas the solution using a pipette immersed in the solution with 5% CO₂, 5% O2, Balance N₂, for 10 minutes (should be a salmon color).
6. Add 100 mg of crystallized Bovine Serum Albumin (BSA).
7. Sterilize the solution by filtration, and gas before storage (do not immerse the pipette).

References

Lawitts, J.A. Biggers, J.D. (1991). Optimization of mouse embryo culture media using simplex methods. J Reprod Fert 91:543-556.

Lawitts, J.A. Biggers, J.D. (1992). Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media. Mol Reprod Dev 31:189-194.

Lawitts, J.A. Biggers, J.D. Culture of preimplantation embryos. In Methods in Enzymology: Guide to Techniques in Mouse Development. P.M. Wasserman and M.L. DePomphilis (eds.) 225:153-164. Academic Press. 1993

Erbach, G.T. Lawitts, J.A. Papaioannou, V.E. Biggers, J.D. (1994).Differential growth of the mouse preimplantation embryo in chemically defined media. Biol. Reprod. 50:1027-1033.

M2 media preparation

M2 medium is conveniently made from stock solutions.

M2 stock solutions

Stock A 10X concentration (expires in 3 months)
Stock B 10X concentration (expires in 2 weeks)
Stock C 100X concentration (expires in 2 weeks)
Stock D100X concentration (expires in 3 months)
Stock E 100X concentration (expires in 3 months)

Preparing M2 stock solutions:

Prepare stocks A, B, C, and D

1. Zero scale with the beaker on it.
2. Add approximately one half of the distilled water necessary for the stock solution.
3. Add the proper amounts of each component stirring occasionally.
4. Add distilled water until scale reads desired grams. (example: Stock A = 200 g, Stock B = 20 g)
5. Filter through a 150 or 250 ml filter system depending on the amount of stock solution made.

Prepare stock E

1. Repeat steps 1-3 from above.
2. Adjust the pH to 7.4 with 0.2 N NaOH.
3. Add distilled water until scale reads desired grams.
4. Filter through a 250ml Filter System.

Preparing M2 from stock solutions

1. Zero scale with the beaker on it.
2. Add the proper amount of each stock solution starting with Stock A and ending at the distilled water. Scale will read 200 g.
3. Add 0.8 grams of Bovine Albumin and let sit until dissolved.
4. Adjust pH to 7.2 – 7.4 if necessary, using 0.2 N NaOH.
5. Filter sterilize with a 22 um filter
6. Measure the osmolality of the medium. It will range between 265 and 280.

M2

*Store stock solutions and M2 in the refrigerator at 4 degrees C.

1 Molar sucrose in M2

Used as an osmotic buffer for embryo cryopreservation.

1. Measure 10ml of M2.
2. Add 3.42 g of sucrose.
3. Mix by gently rock media until sucrose is completely dissolved.

Propylene glycol

1. Measure 8.8 ml of M2.
2. Add 1.2 ml of 1,2-Propanediol.
1. 1,2-Propanediol has a high viscosity.
2. Make sure that all excess Propanediol is removed from the outside of the pipette.
3. After adding the initial amount of Propanediol, allow it to collect at the end of pipette before expelling the remaining Propanediol into the solution.
3. Mix by gently rocking media.

Store at 4°C, discard after 1 week.

MEM medium

1. For a final volume of 200 ml of medium, measure out the following into 174 ml double distilled water:

2. Add 0.2 ml of 1% Phenol red solution.
3. Gas for ten minutes with 5% CO₂, 5% O₂, balance N₂, using a pipette immersed in the solution.
4. Add 600 mg crystallized Bovine Serum Albumin (BSA), and allow to dissolve.
5. 5. Sterilize by filtration, and re-gas before storage.

10X EBSS

200 ml

Calcium dihydrate should be dissolved first in a separate container with millipore H₂O .

**Add the CaCl₂ last after all the other chemicals have dissolved.

Avertin

Components:

2, 2, 2 Tribromoethanol
Tertiary amyl alcohol

Stock solution:

25 g Tribromoethanol in 15.5 ml amyl alcohol.
Store in dark (wrap bottle in foil).

Solution for injection:

1 ml of stock solution to 50 ml of isotonic saline.
Warm to 40ºC, shake well, then store in refrigerator.

Dose:

0.015 ml per gram body weight or 0.4 ml per average adult mouse.

Sperm cryoprotectant

*(Difco has been bought by Becton-Dickinson)

Here are two recipes for making the cryoprotectant (CPA). They differ in how clear the solution will be before filtering; we can detect no difference in function.

Version 1

1. Warm to dissolve the raffinose completely and then dissolve the dehydrated skim milk.
2. Centrifuge at 13,000 x g for 15 minutes
3. Filter the supernatant through a 0.22 micron pore size filter.
4. Aliquot and store frozen.

Version 2

1. Dissolve 18 g dry milk in ultrapure water with a final volume of 300 ml. Dissolve well. The final concentration will be 6%.
2. Centrifuge 1 hour, 12,000 rpm (18,500 x g), 4 deg C.
3. Carefully decant supernatant so that none of the pellet is taken. Using a pipette is a good way to do this. Pouring off the supernatant is not a good method (unless you have very steady hands) because the pellet is not packed very tightly.
4. Take exactly 200 ml of the supernatant. Add 125 ml water and 72 g raffinose. Add water to 400 ml. Stir at room temperature until the raffinose to dissolves.
5. Filter through a 0.2 micron filter. (If you've been sloppy about your decanting step, you might need to pre-filter through a 0.45 micron filter to prevent clogging at this step.)
6. Aliquot using sterile technique and freeze.

Notes

1. The second recipe makes a clearer CPA because centrifuging in the absence of raffinose allows the milk solids to pellet through a much less dense solution. In other words, the first recipe is essentially a density centrifugation.
2. You can make the CPA even clearer by centrifuging longer and/or at higher speeds.
3. Filtering the CPA is easier with a luer-type cartridge that fits on the end of a syringe than with a vacuum unit, because you can generate higher pressures. However, the second recipe results in a CPA that is clear enough to use with a vacuum unit.
4. Remember that CPA is essentially milk and sugar and as such will be a good medium for any bacterial contaminants that you might introduce. Discard any unused solution.

Reference: Nakagata, N. (1996). Use of cryopreservation techniques of embryos and spermatozoa for production of transgenic (Tg) mice and for maintenance of Tg mouse lines. Lab-Anim-Sci 46(2): 236-8.

Gonadotropins

PMSG (pregnant mare serum gonadotropin) is available from:

Sigma Chemical Company
P.O. Box 14508
St. Louis, MO 63178

It is available in 10,000 I.U. vials Cat. #G-4877. Working solutions are made up by adding 400 ml of isotonic saline to a 10,000 I.U. vial of powdered hormone, which is then stirred with a magnetic stirrer for 5-6 minutes. This solution can then be packaged into 12 x 75 mm disposable plastic tubes with caps (we use BIORAD tubes as these are available in colors for coding purposes - PMS and HCG can be kept in different color tubes to avoid mix up) and frozen until needed. Frozen preparations are not kept for longer than three months.

HCG (human chorionic gonadotropin) is also available from Sigma

Sigma Chemical Company
P.O. Box 14508
St. Louis, MO 63178

It is also available in 10,000 I.U. vials Cat. #CG-10. It is diluted in the same way as PMS described above.

For a working solution inject the 10 ml of diluent supplied into the vial of powdered hormone (10,000 I.U.) with a syringe and needle, withdraw the mixed solution and add to 390 ml of isotonic saline. This solution can then be packaged into 12 x 75 mm disposable plastic tubes with caps as for the PMS, but in different color tubes if possible.

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