Freeze cells slowly. Use Ice-cold freezing medium. Once cells are in freezing medium, store cells in box insulated with paper-towels at -20°C until all cells to be frozen are also in freezing medium.

DMSO is poisonous to the cells, because it weakens their membrane. Harsh manipulation of cells in DMSO, or extended contact with DMSO at elevated temperatures will reduce cell viability.

Put insulated box with cells at -80 for at least one overnight period

Finally, transfer cells into liquid nitrogen.

Freezing medium is

80% Normal cell growth medium
10% DMSO, tissue culture grade
10% Fetal Bovine Serum

Gently trypsinize cells from TC dish

Collect cells by gentile centrifugation in clinical centrifuge

Remove supernatant, and gently resuspend cells in cold freezing medium

Immediately put cells into insulated freezing box.

Put Freezing box into -20, and repeat steps 1-4 for any other cells to be frozen. Handle only one dish of cells at a time.

Freeze cells at -80 for at least overnight. (Cells will be viable for up to two years if stored at -80°C)

Freeze cells at liquid nitrogen temperature. - Cells will remain viable up to ten years if stored at -196°C

Thaw cells as quickly and gently as possible, diluting out the DMSO with growth medium and generous (10%) levels of Fetal Bovine Serum.

10ml of Freezing medium should freeze about three 10cm dishes that are 80% confluent.