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Thawing Cyropreserved Embryos



Materials

  • Cryopreserved Embryos — Millipore cat. no. CRY-BL6-8
  • 2 X 30 mm dishes (or similar) each containing approximately 1.0 mL of holding media (FHM, M-2)
  • 1 X 30 mm dish (or similar) containing 3 drops of KSOM with amino acids
    • Drops should be 50 µL under light mineral oil
    • Media should be equilibrated at 37°C and 5% CO2 overnight
  • Sterile Scissors
  • Timer
  • Graduated cylinder or similar vessel tall enough for the straw containing embryos to be immersed
  • 37°C water bath
  • Transfer pipettes
  • Kimwipes® Wipes
  • Rack capable of holding the straw by the ends such that the embryo column is suspended in air not touching the rack.




Protocol

  1. Remove straw from nitrogen container by handle. Place on rack or between two racks such that the straw is held in a horizontal position and the column of liquid containing the embryos is not touching a solid surface.
  2. Thaw straw for two minutes in air. Remove handle (handle easily pulls away from straw) and gently wipe straw with a Kimwipe to remove condensation. Be certain that the straw has thawed (no ice crystals visible).
  3. Shake the straw as you would a fever thermometer by holding the sealed end and shaking down towards the plug end. The intention is to mix the column containing the embryos into the sucrose containing column. (Vigorous shaking is called for here and will not harm the embryos). The result should be a continuous column of liquid with an air space between the liquid and the sealed end.
  4. Incubate the straw at 37°C for precisely three minutes, in the vertical position, holding the plug end up. The embryos will settle to the bottom of the now continuous liquid column. This step allows the cryoprotectant to efflux from the embryos.
  5. Remove the straw and cool to 20°C by immersing in water contained in grad cylinder for five seconds.
  6. Snip off the sealed end (there will be an air space between the sealed end and the liquid column).
  7. Hold the snipped end over a 1.0 mL drop of holding media in 30 mm dish.
  8. Cut the plug end (you may be cutting into the liquid column just below the plug but the embryos would have settled to the bottom of the liquid column during 37°C incubation). As soon as you have cut the plug end the liquid column will be released into the drop of media.
  9. Remove the dish to the microscope. With transfer pipette, transfer the embryos to the second drop of holding media, and then through 2 drops of KSOM. When all of the embryos have been placed in the final (third drop) of KSOM, remove dish to incubator (37°C with 5% CO2).

    Note: If embryos are viewed in the first drop of holding media they will appear somewhat shriveled, this is normal as the embryos recover from the cryoprotectant.

    Note: Plug end is sealed, and pushed into handle. Plug may be partially obscured by label.

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