It is best to freeze cells that are growing rapidly. With adherent cells, it is easiest to set up 100 mm dishes, give them fresh medium the day before you freeze them, and freeze them just as they become confluent.

If you are working with suspension cells, make sure that they are growing vigorously before you freeze them.

1. Trypsinize the cells if necessary, and then spin them down.

Resuspend them in one half the volume that you want to end up with.

In other words, use 0.5 ml per 100 mm dish you are using.

The cells should be resuspended in DMEM containing 20% fetal calf serum.

Put the tube in an ice bucket and let the cells cool.

2. Now, add the DMSO to the cells slowly. This step is accomplished by adding to the resuspended cells an equal volume of DMEM containing 20% fetal calf serum and 20% DMSO.

This should be done dropwise and the cell suspension should be mixed by swirling after the addition of each drop.

The reason to do this slowly is to avoid osmotic shock and to minimize the heating that occurs when DMSO is added to water. When you are finished, you should have the cells suspended in DMEM containing 20% fetal calf serum and 10% DMSO with all the cells from a single 100 mm dish in 1 ml.

3. Chill the cells on ice, in the centrifuge tube, for exactly 30 min. Then transfer them to freezing vials which have been pre-chilled in the freezer in the plastic rack, which has also been pre-chilled in the freezer. Use 1 ml per vial.

4. Put the vials in a well insulated box that has also been pre-chilled in the freezer. Insulation is best accomplished with cotton.

Then put the box in the -70°C freezer overnight, for slow cooling, and then put them into liquid nitrogen.

Cells will store forever in liquid nitrogen. Storage at -70°C is riskier.

Thawing.

Cells should be thawed rapidly and then diluted slowly into warm growth medium.

The best way to do this is to transfer the contents of the thawed vial to a 50 ml centrifuge tube and then add--dropwise, with continual swirling--10 ml of warm medium.

Then put the cells on a 100 ml dish. This way the cells don't suffer osmotic shock.