Sack: Freezing CHO cells

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Freezing CHO Cells
Christina McGee 4-5-2012

  1. Grow 10 cm dish to 50-90% confluency in T-75 flask
  2. Digest cells with 3 ml Trypsin/EDTA, wait for cells to detach.
  3. Add 7 ml media, transfer to 15 ml sterile tube.
  4. Centrifuge 250 G/5 min.
  5. Remove supernatant. Gently resuspend cell pellet in 2 ml freezing medium*.
  6. Transfer 0.5 ml to 4 freezing vials labeled with name of cell line, date and your initials.
  7. Place vial in freezing box in -80°C freezer.
  8. Transfer to liquid nitrogen 1-4 days later.
  9. Record location of cells in Liquid N2 excel spreadsheet

  • Freezing medium: Cell medium (F-12 or DMEM/F-12) without selection agents or antibiotics plus 20% FBS and 10% DMSO (dimetylsulfoxide, sterile).