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Mouse Embryonic Stem Cell Core Facility
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Cryopreservation of Mouse ES Cells


  • Freezing vials (1 or 2 ml size)
  • Styrofoam or iso-Propanol container
  • -80° freezer
  • Liquid Nitrogen Storage Tank

M15 or ES Cell Culture Media:

  • 82% high Glucose DMEM (Invitrogen, catalog number: 12100-061)
  • 15% Fetal Calf Serum (ES Cell Tested)
  • 1% 200mM L-Glutamine (Invitrogen, catalog number: 25030-081)
  • 1% Penicillin Streptomycin (Invitrogen, catalog number: 15140-122)
  • 1% 10mM ß-mercaptoethanol (Sigma, catalog number: M 7522)
    (7.5ul ß-mercaptoethanol + 10ml PBS)

ES Cell Freezing Media:

2X Strength:

  • 60% DMEM high Glucose (Invitrogen, catalog number: 12100-061)
  • 20% FCS (ES Cell tested)
  • 20% DMSO (Sigma, catalog number: D-2650)

Working Solution (1X Freezing Media):

  • 1 part 2X freezing media + 1 part M15


  1. Feed cells 2 hours prior trypsin treatment.
  2. Rinse plate twice with PBS.
  3. Trypsinize cells for 6-10 minutes at 37°C.
  4. Place an equal volume of ES Cell Media as Trypsin and pipette vigorously in order to disrupt any cell clumps.
  5. Using ES Cell Media, bring volume up to 10mls and place in a sterile 15ml conical tube.
  6. Using a hemocytometer count cells to determine total number of cells in tube.
  7. Spin at 600rpm for 5 minutes to pellet cells.
  8. Resuspend cell pellet in enough freezing media to make a 1 x 107 cells per ml suspension.
  9. Place one ml cell suspension per cryovial.
  10. Allow to freeze overnight in Styrofoam container at -80°C.
  11. Place in LN2 storage only after overnight storage at -80°C.