Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Published 11/18/2011
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Summary

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

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Mochida, K., Hasegawa, A., Taguma, K., Yoshiki, A., Ogura, A. Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification. J. Vis. Exp. (57), e3155, doi:10.3791/3155 (2011).

Abstract

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4°C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.

Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

Protocol

The overall scheme of the experiment is shown in Fig. 1.

1. Reagent Preparation

  1. Make the base medium (here in known as PB1) in a 100 ml glass bottle according to the following chart below:
      M.W. mM mg/100ml
    NaCl 58.4 136.98 800.0
    KCl 74.6 2.68 20.0
    KH2PO4 136.1 1.47 20.0
    Na2HPO4.12H2O 358.14 8.04 288.1
    MgCl2,6H2O 203.3 0.49 10.0
    Glucose 180.2 5.56 100.0
    Na pyruvate 110 0.33 3.6
    CaCl2,2H2O 147 0.9 13.2
    Penicillin G     6.0 (approx)
  2. Make the Ficoll-sucrose (FS) solution :
    1. Add the following chemicals to 14 ml of PB1 solution in a 50 ml tube.
      Ficoll 70 6.0 g
      Sucrose 3.424 g
      BSA 42.0 mg
    2. Mix Ficoll 70 and sucrose in PB1 solution and shake well until completely dissolved.
    3. After checking complete dissolution above, add BSA on the surface of the solution and keep it at 4oC until BSA is completely dissolved (> 4 hours or overnight).
  3. Make the equilibration solution (EFS20) and the vitrification solution (EFS40) in a 50 ml tube:
    • EFS20: 20%(v/v) ethylene glycol, 24% (w/v) Ficoll, and 0.4 mol/L sucrose in PB1 with BSA
    • EFS40: 40%(v/v) ethylene glycol, 18% (w/v) Ficoll, and 0.3 mol/L* sucrose in PB1 with BSA
      *For embryos from the BALB/c or ICR strain, increase the concentration of sucrose to 0.9 mol/L sucrose because they are more sensitive to cryodamage6.
      EFS20 solution EFS40 solution
    Ethylene glycol 1 ml 2 ml
    FS solution 4 ml 3 ml
  4. Sterilize the solution by filtration using a 0.45 μm filter. Make aliquots in 5 ml polystyrene tubes and store at 4°C. They can be used for about 6 months.
  5. Preparation of embryo thawing solution containing 0.75 M sucrose (TS1)
    1. Dissolve 7.7 g of sucrose in PB1 (prepared in step 1.1) and bring the total volume to 30 ml. Mix by gentle shaking until sucrose is completely dissolved.
    2. Add 90 mg of BSA onto the surface the solution and leave it stand until completely dissolved.
    3. Sterilize the solution by filtration.
    4. Aliquot and store at 4°C. They can be used for about 1 month.

    Note: TS1 can also be prepared from commercially available M2.
    1. Dissolve 7.7 g of sucrose in M2 and bring the total volume to 30 ml.
    2. Mix by gentle shaking until sucrose is completely dissolved.
    3. Sterilize the solution by filtration.
    4. Aliquot and store at 4oC. They can be used for about 1 month.
  6. TS2 (thawing solution containing 0.25 M sucrose):
    1. Dilute 10 ml TS1 with 20 ml volume of PB1 or M2, as appropriate.
    2. Aliquot and store at 4°C. They can be used for about 1 month.

2. Vitrification of 2-cell Mouse Embryos

  1. Prepare 2-cell mouse embryos by natural mating or conventional in vitro fertilization techniques.
  2. Aliquot 50 μl EFS40 into a cryotube. Hereafter, perform the rest of the vitrification procedure at the room temperature.
  3. Aliquot 50 μl of EFS20 on the bottom of a 35 mm or 60 mm plastic Petri dish.
  4. Transfer up to 30 embryos to EFS20 using a glass capillary with only the minimum amount of the culture medium. Start a timer for 2 min.
    Note: Place embryos on the bottom of the drop. This makes efficient dehydration of embryos. Check the morphology of embryos by a stereomicroscope. Dehydrated embryos show a shrunken morphology, as shown in Fig. 2A. If they are not dehydrated enough, wait for more 1 or 2 min.
  5. At about 1.5 min, pick up the embryos from the EFS20 drop with only the minimum amount of the solution. Transfer them to EFS40 in the cryotube prepared in step 2.1. Note: For the best results, adjust the timing of picking up embryos so that all embryos can be transferred into EFS40 at around 2 min.
  6. Wait for 1 min.
  7. Put the cryotube directly into liquid nitrogen (LN2).

3. Thawing Vitrified 2-cell Mouse Embryos

  1. Before thawing, prepare a dish with 10 μl drops of embryo culture medium for recovered embryos (see step 3.13). Cover the dish with oil and place in a CO2 incubator until use. Note: Although any media for routine embryo culture may be used in this experiment, we recommend media with higher osmolarity such as M16 medium.
  2. Warm TS1 to 37°C.
  3. Put on a face mask and cryogloves. Open the LN2 tank and retrieve a cryotube containing embryos.
  4. Quickly open the top of the tube and discard LN2. Wait for 30 sec to prevent TS1 from freezing at the next step.
  5. Use a 1000ul pipette to add 850 μl of TS1 (37°C) into the tube and mix the solution by gentle pipettings (about ten times in 25 seconds) until the solution is evenly dissolved.
  6. Transfer the entire volume of the tube to a plastic 60 mm Petri dish (or a watch glass) (Fig. 3A). Note: Embryos should be handled at room temperature (22-25°C) until they are placed in a CO2 incubator at Step 3.14.
  7. Start a timer for 3 min.
  8. At around 2 min in TS1, gently shake the dish until the medium containing embryos is spread over the surface of the dish (Fig. 3B). Note: This will help the embryos go down to the bottom because they are floating near the surface when transferred to TS1.
  9. Put three 50 μl drops of TS2 onto the dish (Fig. 3C).
  10. After 3 min in TS1, check the morphology of embryos by a stereomicroscope; they should be slightly shrunken. See the morphology of embryos in earlier (Fig. 2B) and later (Fig. 2C) in TS1. Note: If the embryos are still swollen, keep them in TS1 for more 1 to 3 min.
  11. Pick up the embryos and transfer them to the first drop of TS2 (Fig. 3D). Start a timer for 3 min
  12. After 3 min, transfer embryos to the second drop and then to the third drop (Fig. 3E).
  13. Transfer the embryos to the culture medium prepared at Step 3.1. The embryos are in the shape shown in Fig. 2D.
  14. Place dish in a CO2 incubator. About 10 min later, transfer embryos to the next drop of culture medium to wash out sucrose that has been carried over from TS2.
  15. Continue culture in a CO2 incubator until embryo transfer.
    Note: Transfer embryos to the oviducts of recipient females on the day of thawing. Long culture in vitro may decrease the viability of embryos in some strains of mice.

4. Representative Results:

In vitro- and in vivo-development of embryos after thawing is presented in Tables 1 and 2. The advantages of this protocol are the high survivability of embryos after thawing and its broad applicability to different strains of mice.

Strain Total No. of tubes No. of embryos vitrified Recovered (No. (%)) Morphologically normal (No. (%)) Development to blastocysts (No. (%))
C57BL/6J 20 400 397 (99) 394 (99) 342 (87)
BALB/cA 15 300 296 (99) 282 (95) 238 (84)
ICR 24 480 474 (99) 443 (93) 398 (90)

Table 1. In vitro-development of vitrified-thawed embryos in common mouse strains

Condition of embryos No. of recipient females No. of embryos transferred Implantation sites (No. (%)) Live offspring (No. (%))
Fresh 12 180 141 (78.3) 110 (61.1)
Vitrified 16 242 202 (83.5) 125 (51.7)

Table 2. In vivo-development of vitrified-thawed embryos in C57BL/6J mice.

Figure 1
Figure 1. The overall scheme of the experiment including equilibration, vitrification, and thawing of embryos.

Figure 2
Figure 2. The morphology of embryos at each step of thawing.

Figure 3
Figure 3. Thawing procedure of embryos. All procedures are performed at room temperature. (A), Add 850 μl of TS1 (37°C) into a cryotube and transfer the entire volume of the solution in the cryotube onto a plastic dish. At this time, the embryos look swollen as shown in Fig. 2B. (B), Spread the solution over the surface of the dish by gentle shaking. (C), Place three 50 μl drops of TS2 on the plastic dish. (D), Transfer the embryos to the first drop of TS2. After 3 min, the embryos look shurunken as shown in Fig. 2C. (E), Transfer them serially into the remaining TS2 drops and then to the culture medium.

Discussion

Since the first report of mouse embryo vitrification by Rall and Fahy in 19852, several technical improvements have been made to increase the survivability of embryos after thawing. One of the most successful modifications was achieved by use of ethylene glycol as a cryoprotectant because of its low toxicity and high membrane-permeability. Such advantages enable us to handle freezing embryos at room temperature4; other vitrification methods require embryo handing at cooler temperatures and are not always applicable to some strains of mice including BALB/c6. The first ethylene glycol-based vitrification was developed by Kasai et al. in 19905,7. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. Therefore, the vitrification method described here may be applicable to many laboratories using mice as research models. The same method can also be used for mouse embryos at the morula and blastocyst stages8 and rat embryos9. However, we have recently found that the quality of cryotubes may affect the survival rates of embryos after freezing-thawing. Thus, it is essential to examine the inside surface of the cryotubes for its smoothness before vitrifying embryos (e.g.; see at Table of specific reagents and equipment).

Vitrification methods have many advantages over conventional slow freezing methods, but they inherently have a disadvantage in respect to transportation purpose. As vitrified embryos should be kept at below -120°C to maintain their viability, dry shippers are generally used for their safe transportation. Dry shippers are heavy and bulky, and their round-trip is expensive, especially for international transportation. We are now currently developing a new vitrification method by which vitrified embryos can be stored at dry-ice temperature (about -80°C) for at least 7 days10. This method should be the vitrification of the next generation.

Disclosures

We have nothing to disclose.

Acknowledgements

This study was conducted in cooperation with the National BioResource Project, the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Materials

Name Company Catalog Number Comments
Bovine serum albumin Merck & Co., Inc. 12657
Ethylene glycol Wako Pure Chemical Industries, Ltd. 058-00986
Ficoll 70 GE Healthcare 17-0310-10
Glucose Wako Pure Chemical Industries, Ltd. 041-00595
NaCl Wako Pure Chemical Industries, Ltd. 191-01665
KCl Wako Pure Chemical Industries, Ltd. 163-03545
KH2PO4 Wako Pure Chemical Industries, Ltd. 169-04245
Na2HPO4·12H2O Wako Pure Chemical Industries, Ltd. 500-04195
MgCl2 ·6H2O Wako Pure Chemical Industries, Ltd. 135-00165
CaCl2 ·2H2O Wako Pure Chemical Industries, Ltd. 031-00435
Penicillin G Sigma-Aldrich P-4687
Sodium pyruvate Sigma-Aldrich P-8574
Sucrose Wako Pure Chemical Industries, Ltd. 196-00015
M16 medium Sigma-Aldrich M7292
M2 medium Sigma-Aldrich M7167
Cryotube Sumitomo Bakelite Co., Ltd. MS-4501 “Cryogenic Vial”

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References

  1. Whittingham, D. G., Leibo, S. P., Mazur, P. Survival of mouse embryos frozen to -196° and -269°C. Science. 187, 411-414 (1972).
  2. Rall, W. F., Fahy, G. M. Ice-free cryopreservation of mouse embryos at -196°C by vitrification. Nature. 313, 573-575 (1985).
  3. Yoshiki, A. The mouse resources at the RIKEN BioResource center. Exp. Anim. 58, 85-96 (2009).
  4. Mochida, K., Ogura, A. Cryopreservation of embryos in laboratory species. J. Mamm. Ova. Res. 27, 87-92 (2010).
  5. Kasai, M. A simple method for mouse embryos cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J. Reprod. Fert. 89, 91-97 (1990).
  6. Dinnyes, A., Wallace, G. A., Rall, W. F. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods. Mol. Reprod. Dev. 40, 429-435 (1995).
  7. Kasai, M., Mukaida, T. Cryopreservation of animal and human embryos by vitrification. Reprod. Biomed. Online. 9, 164-170 (2004).
  8. Miyake, T. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method. Theriogenology. 40, 121-134 (1993).
  9. Han, M. S., Niwa, K., Kasai, M. Vitrification of rat embryos at various developmental stages. Theriogenology. 59, 1851-1863 (2003).
  10. Jin, B. Equilibrium vitrification of mouse embryos. Biol. Reprod. 82, 444-450 (2010).

Comments

14 Comments

  1. Please, one question about the medium EFS²0 and EFS40:
    Are they prepared simply using the proportions described in the table EG/FS 1:4 and EGFS ²:5 (table), or is it the another formula described in the text (EFS²0: ²0% (v/v) etthylene glycol, ²4% (w/v) Ficoll and 0.4mol/L sucrose in PB1 with BSA. Thanks in advance .Cleide from Brazil.

    Reply
    Posted by: Cleide S.
    May 17, 2012 - 2:23 PM
  2. Thank you for your interesting.
    The first, we prepare FS solution consist of 30% (w/v) Ficoll and 0.5 mol/L sucrose in PB1 with BSA. And mix with ethylene glycol 1:4 or ²:3 for preparation of EFS²0 and EFS40, respectively. Finally, EFS²0 consists with ²0% (v/v) ethylene glycol, ²4% (w/v) Ficoll and 0.4mol/L sucrose in PB1 with BSA.
    Keiji from Japan

    Reply
    Posted by: Keiji M.
    May 18, 2012 - 12:44 AM
  3. Thanks Keiji for your response.
    I have another question: what embryo culture medium did you use until cryopreservation?
    You have very good results.
    congratulations
    Cleide

    Reply
    Posted by: Cleide S.
    May 18, 2012 - 3:28 PM
  4. I have always used CZB medium (Chatot CL, et al. 1989, J. Reprod. Fertil. 86:679-688) containing 5.6 mM glucose, 0.1 mg/ml PVA, and 3.0 mg/ml bovine serum albumin after IVF for around ²0 hrs and after cryopreservation until embryo transfer.
    The data in table 1 were obtained with CZB medium.
    I also recommend you to use KSOM medium instead of CZB medium.

    Keiji

    Reply
    Posted by: Keiji M.
    May 20, 2012 - 9:53 PM
  5. Ok Keiji.
    Thank you very much.
    best regards
    Cleide

    Reply
    Posted by: Cleide S.
    May 21, 2012 - 9:56 AM
  6. Keiji,
    I'm very interested in your protocol. I've just a question about the preparation of the PB1 solution. Do you just dissolve all the components in the order of your list in 100mL distilled H²0 or do you readjust the pH?
    Thanks in advance.
    Maryline from Belgium

    Reply
    Posted by: Maryline K.
    July 6, 2012 - 4:49 AM
  7. Thank you for your interesting.

    About PB1 preparation , you can also find the protocol in the book, "Manipulating the Mouse Embryo" as modified D-PBS.
    We can prepare this solution to dissolve all components in the order of our list.

    However, usually we prepare PBS (-) solution first, which consists of NaCl, KCl, Na²HPO4 and KH²PO4.
    Then, after preparation of x100 concentrated MgCl² and CaCl² solutions, respectively, each 0.1 ml solution are slowly added in 98 ml of PBS(-) solution with gently mixing.
    Because this PBS(+) solution can be stored in refrigerator for several months.
    Before using, we dissolve other reagents, glucose, Na pyruvate and penicillin G.
    This PB1 solution can be used for at least a few weeks by storing in refrigerator.
    In the other hand, we can buy the powder or tablet of PBS(-) from some companies.

    And we do not adjust the pH.

    I hope this coment will be a help for you.

    Reply
    Posted by: Keiji M.
    July 8, 2012 - 11:05 PM
  8. Thank you very much! Your coment is really helpful.
    Maryline

    Reply
    Posted by: Maryline K.
    July 9, 2012 - 2:36 AM
  9. Hello,
    I wanted to try this method using 8-cell embryo. I noticed that the freezing media the PB1 is with BSA. How much BSA do you add?

    Thanks,
    Agnes
    Thanks,

    Reply
    Posted by: Anonymous
    April 5, 2016 - 5:30 PM
  10. Thank you for your interesting.

    PB1 contains 3 mg/ml of BSA. Finally, vitrification solution (EFS40) contains 1.26 mg/ml of BSA.

    And this method is effective for the embryos at each stage from 1-cell to early blastocyst include 8-cell stage.

    Reply
    Posted by: Keiji M.
    April 6, 2016 - 8:30 PM
  11. Hello,
    I tried the procedure but I found out that it is very hard to see the embryos after transferring them in EFS20. They floated and changed shape. I lost half the number of the embryos that I collected. Is there any practical tip that you can tell me to avoid this problem?
    Also, could I harvest the embryos in M2 and freeze them using you freezing media?

    Thanks again.

    Reply
    Posted by: Anonymous
    April 8, 2016 - 1:22 PM
  12. Thank you for your interesting.

    If you can release embryos with small volume of medium near the bottom of EFS20 solution, you can find all embryos in small area. And we also recommend that it becomes easy to discover embryos at the surface of EFS20 solution by control of the light with the adjustment lever.

    The developmental ability of embryos will decrease at outside of incubator. However, the embryos must be left outside of incubator more than 10 min., the medium contains HEPES (ex. M2, HEPES-KSOM) or PB1 solution should be used.

    Keiji

    Reply
    Posted by: Keiji M.
    May 2, 2016 - 1:13 AM
  13. Hi,

    I think there is a mistake in the step 1.2.1: Add the following chemicals to 14 ml of PB1 solution in a 50 ml tube.
    Shouldn't it be 20ml?

    Thanks,
    Tony

    Reply
    Posted by: Tony N.
    April 28, 2016 - 4:16 AM
  14. Hi, Tony

    You are right.
    Total volume will be 20 ml.
    The conical tube of 50 ml is useful for preparation of this solution.

    Thank you.

    Keiji

    Reply
    Posted by: Keiji M.
    May 2, 2016 - 1:20 AM

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