Embryo Cryopreservation by Slow Freezing

This procedure is the one that has been used in The Jackson Laboratory for many years. It is based on the protocol originally described by Whittingham et al. (1972). See also Mobraaten (1999). Other methods of slow freezing are reported by Schmidt et al. (1987), Liu et al. (1993a), Dinnyes et al. (1995), and Shaw et al. (1995, 2000a). It is important to note that if straws are substituted for vials, or if a different controlled-rate freezer is used in the procedure described below, adjustments may be needed in timing and freezing and thawing rates due to the changes in heat transfer rate.

Materials

Animals

  • Pseudopregnant females at 0.5 or 2.5 dpc for oviduct or uterine transfer (see Chapters 3 and 6)
  • Superovulated donors at 2.5 dpc

Cryogenic Equipment

  • CryoMed 1010 programmable freezing controller (Forma Scientific)
  • Cryotubes (Nunc 12-565-170N)
  • Forceps (large)
  • Gloves and goggles for handling LN2
  • Liquid nitrogen (LN2)
  • LN2 containers (Dewar flasks) for benchtop work
  • Permanent markers or other means of labeling
  • Storage goblets and canes or boxes
  • Tongs with long handles to manipulate samples

General Equipment for Embryo Handling

  • Culture dishes, sterile (e.g., Falcon 3001, 3004, 3037)
  • Embryo-handling pipette
  • Flushing needle
  • Microdrop cultures
  • Microscopes with transmitted and reflected light
  • Pasteur pipette
  • Selectapette (Clay-Adams) (optional)
  • Surgical instruments
  • Syringes, 1 ml with 26- and 30-gauge needles
  • Watch glass or depression slide

Reagents

D-PBS supplemented with 1000 mg/liter dextrose (e.g., Sigma G6152), 36 mg/ml sodium pyruvate, penicillin G (0.075 mg/ml), streptomycin sulfate (0.05 mg/ml) Containing 3 mg/ml of embryo-tested bovine serum albumin (BSA) (used for embryo handling)

  • BSA-free (used for preparation of 2 M DMSO cryoprotectant)

Dimethyl sulfoxide (DMSO) m.w.78.13 (e.g., Sigma D8779 or D2650)
To make up 10 ml of 2 M DMSO, measure out 1.56 g of DMSO (~1.42 ml) in a tube and add 8.58 ml of BSA-free D-PBS.

Embryo-tested light mineral oil (e.g., Sigma M8410)

Ethanol, 95%

KSOM-AA medium

CAUTION: See Appendix 2 for appropriate handling of materials marked with

Procedure

Freezing

  1. Precool the freezing machine to -6ºC.
  2. Collect eight-cell-stage embryos in the morning of 2.5 dpc as described  using D-PBS supplemented with 1000 mg/ml dextrose, 36 mg/liter sodium pyruvate, 3 mg/ml BSA, and antibodies.
  3. Transfer embryos into a 2-ml cryotube containing 0.1 ml of D-PBS and place the tube on ice until all embryos have been collected and are ready for freezing.  The number of embryos placed into one cryotube depends on their expected viability and should be sufficient for transfer into at least two recipients (e.g., around 30 embryos).
  4. Gently add 0.1 ml of protein-free D-PBS containing 2 M DMSO at 0ºC to bring the final concentration of DMSO to 1 M.  Equilibrate at 0ºC for at least 30 min.
  5. Transfer the cryotubes to a salt and ice bath maintaining -6ºC (or to the freezing machine at -6ºC).  Equilibrate for 2 minutes.
  6. Seed the contents of the cryotube by touching the surface of the medium with an ice crystal from the tip of a Pasteur pipette.  (Draw a small amount of PBS into the tip of the Pasteur pipette by capillary action, and put it inside a glass test tube that is partially immersed in a salt and ice bath maintained at -10ºC).  Use a separate pipette for each seeding.  Seeding starts ice nucleation of the medium with minimal damage to the embryos caused by the formation of ice crystals.  Cap the cryotubes and transfer them to a controlled-rate freezer precooled to -6ºC.
  7. Cryotubes (or straws) also can be seeded by touching the outer surface of the cryotube leveled with the meniscus of cryoprotectant, away from embryos, with a  metal object (e.g., long-handled forceps) precooled in LN2.
  8. Lower the temperature in a controlled-rate freezer at the rate of 0.5ºC per minute to -80ºC.  If the freezing machine does not have a temperature display, monitor the temperature using a thermocouple inserted into a control cyrotube containing 0.2 ml of 95% ethanol.
  9. When the temperature reaches -80ºC, transfer the cryotubes to LN2.  It mahy be most convenient to place them into a small LN2 flask first and then transfer the samples to a large LN2 container for long-term storage.  Record strain information, number of embryos, location, etc.

Thawing

  1. Remove the cryotube from the LN2 and allow it to warm at ambient temperature until all ice crystals have thawed (usually 12-15 minutes).
  2. When completely thawed, slowly add 0.8 ml of D-PBS in dropwise fashion to dilute the DMSO.
  3. Transfer the contents of the cryotube to a dish, embryological watch glass, or depression slide and observe the embryos.  A 1-ml Selectapette (Clay-Adams) may be used.
  4. When the embryos through several drops of pre-equilibrated KSOM-AA medium and culture them for a few hours or overnight in microdrops covered with embryo-tested oil as described in Protocol 4.5. An alternate way of embryo culture in glass tubes is described at: http://www.jax.org/resources/documents/cryo/slow.html.
  5. Transfer the embryos into the oviduct or uterus of pseudopregnant females.

Comments

All freezing parameters described above are for the CryoMed 1010 programmable freezing controller (Forma Scientific) used in The Jackson Laboratory. Many varieties of controlled rate freezers are commercially available (see partial list in Chapter 17) and may be divided into two large groups: alcohol-based and LN2-based. Alcohol-based freezers are generally less expensive than LN2-based freezers and can be used reliably and cost-effectively for embryo cryopreservation in insemination straws sealed glass ampules. However, they may not be the best choice for embryo cryopreservation in cryotubes because the cryotubes do not provide a tight seal to prevent alcohol penetration inside the vial. Liquid nitrogen-based freezers are a better option for embryo cryopreservation in plastic cryotubes.