FreezingandThawingofMammalianCellLines

For long term storage of myeloma cells, hybridoma cells, T cells, and other mammalian cell lines in liquid nitrogen, and restoring them in culture.

Freezing

Preparation

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.

Freezing

1. 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.

2. Place vials upright in a styrofoam box and cover well. Place in a -70 °C for 24 hours.

3. Place vials in a wand and put in a liquid nitrogen container.

Thawing

Preparation

1. Warm water bath to 37 °C.

2. 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.

3. Make your growth media for your new culture. For monoclonals this would be DMEM with 20% FBS and penicillin-streptomycin.

Thawing

1. Take your cells out of nitrogen storage and thaw rapidly by swirling in the 37 °C water bath.

2. 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 the FBS.

3. Centrifuge on "3" for 10 min. in a clinical centrifuge.

4. 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.