Freezing and Thawing of Mammalian
For long term storage of myeloma cells, hybridoma cells,
T cells, and other mammalian cell lines in liquid nitrogen, and restoring
them in culture.
Cells are to be frozen in liquid nitrogen, so make sure
your canisters are relatively full of nitrogen and you have room. Cells
should be healthy (>90% viability) and growing in log phase. You will
also require sterile 1 mL cryo-vials; they have a screw-top and rubber seal
to keep the nitrogen out.
- Count cells in a hemocytometer. Centrifuge 10 mL of cells
on "2" in a clinical centrifuge for 10 minutes and resuspend
in freezing medium (10% DMSO, 20% FCS, 70% media that you used to grow
the cells such as RPMI or DMEM) at a concentration of 2 X 10e6 cells/0.5
mL freezing medium. Aliquot in cryo-vials 0.5 mL/vial.
- Place vials upright in a styrofoam box and cover well.
Place in a -70 °C for 24 hours.
- Place vials in a wand and put in a liquid nitrogen container.
- Warm water bath to 37 °C.
- Place 10 mL media (RPMI, DMEM) in a sterile 15 mL centrifuge
tube. Layer 2 mL FBS to the bottom of the tube, slowly, so that you can
see two layers.
- Make your growth media for your new culture. For monoclonals
this would be DMEM with 20% FBS and penicillin-streptomycin.
- Take your cells out of nitrogen storage and thaw rapidly
by swirling in the 37 °C water bath.
- Sterilize the outside of the vial with 70% ethanol, bring
in to the culture hood and add slowly to the top of the layered media you
prepared. The cells should fall to the interface between the media and
- Centrifuge on "3" for 10 min. in a clinical
- Pipet of the supernatant and resuspend in the growth
media of choice you prepared earlier. Pipet into a T25 and place in a CO2 incubator for growth of your new culture.
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