Marek: Freeze-down/Thaw

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This short protocol describes how to freeze down and thaw (or bring up) mammalian cells (e.g. HeLa, KEK293, CHO, Jurkat T cells).


List reagents, supplies and equipment necessary to perform the protocol here.

  • 10-50 ml centrifuge tube
  • cryo tubes
  • DMSO, Tissue culture (TC)-tested (e.g. Sigma, D2650)
  • Cell culture medium (depends on cell line/cells)
  • Trypsin-EDTA (e.g. from PAA, L11-004) - for adeherent cells
  • Freeze-down medium
    • 6-10% DMSO
    • Cell culture medium (I recommend to use at least 20% FCS and you can use 100% fetal calf serum (FCS) instead of cell culture medium)
  • Basic TC centriguge (0 - 2500 rpm)
  • Freeze-down box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )

Freeze-down (suspension cells)

  1. Centrifuge at 80-100 x g for 4-5 min at RT.
  2. Suck out the medium.
  3. Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
  4. Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.
*Leave over-night at -80 gr.C.
*You can store cells at -80 gr.C for up to 6 months.
*Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).

Freeze-down (adherent cells)

  1. Wash cells with PBS.
  2. Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)
  3. Observe cells under microscope until these are rounded.
  4. Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.
  5. Centrifuge at 1000-1200 rpm for 4-5 min at RT.
  6. Suck out the medium.
  7. Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
  8. Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).


  1. Keep frozen cells on dry ice until ready for thawing.
  2. Swirl a vial with frozen cells in 37gr.C water bath and remove just while a sliver of ice remains.
  3. Spray tube with 70% ethanol for sterilization.
  4. Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
  5. Fill centrifugation tube with culture medium: at least 10 ml total volume.
  6. Centrifuge for 5 min at 80-100 x g (RT).
  7. Resuspend pelleted cells in appropriate volume of culture medium and culture o/n in CO2 incubator.
  8. Optional: Exchange medium next day.


  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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