Cell Freezing Medium

Protocol 1

Serum-Free Cell Freezing Medium: 1 % (v/w) sericin, 0.5 % (v/w) maltose, 0.3 % (v/w) proline, 0.3 %(v/w) glutamine and 10 % DMSO in PBS.

Regular Cell Freezing Medium: 10% DMSO, 20% FCS, 70% media

Freezing

1. Spin down cells at 1000 rpm at 4 oC.

2. Resuspend at 1 × 10^6 cells/ml in a freezing medium

3. Dispensed into cryovials.

4. Put on ice for 5 min and then place at −80 oC freezer for 1 day.

5. Put cryovials into liquid N2

Thawing

1. Retrieve cryovials from the LN2 freezer and immediately place in a 37 oC water bath.

2. Spin down cells at 1000 rpm cells are thawed.

3.Optional: Resuspend cells in 1 ml of PBS and spin again

4. Add normal cell culture medium and seed cells into culture dish or plate.

Protocol 2

Freezing

Preparation

Cells should be healthy (>90% viability) and growing in log phase.

Sterile 1 mL cryo-vials.

Freezing

Count cells in a hemocytometer.

Spin down cells at 1000 rpm for 1 min.

Resuspend cells in freezing medium (10% DMSO, 20% FCS, 70% media in RPMI or DMEM) at a concentration of 4 X 10e6 cells/1 mL freezing medium.

Aliquot in cryovials 1 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.

Thawing

Preparation

Warm water bath to 37 °C.

Place 5 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 growth media for  new culture. For monoclonals this would be DMEM with 20% FBS and penicillin-streptomycin.

Thawing

Take 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.

The cells should fall to the interface between the media and the FBS.

Spin for 10 min at 1000 or 3000 rpm.

Pipet out supernatant and resuspend cells in the growth media of choice.

Pipet cells into a T25 and place in a CO2 incubator for growth.