A. Freezing Cells -  From the class session, you know that the objective in freezing cells is to do so relatively slowly (i.e. approximating 1C per minute) in the presence of the cryoprotectant DMSO, and to minimize the formation of ice crystals inside the cytoplasm. At that rate of freezing, most of the ice crystals form outside of the cytoplasm. However, as the ice crystals form outside of the cell, the intracellular water moves to the outside leaving the cells in a plasmolyzed state. Without the cryoprotectant, which binds water keeping a sufficient amount inside the cells to maintain cell viability, the cells would not survive the process.
1. Remove 2 x 10^6 cells from the suspension of cells remaining after completing the exercise above and centrifuge, at 600 rpm for 5 min.
2. Aspirate off  the supernate and replace with 2 ml of 1x cryoprotective medium (EBME₈₅, DMSO₅, FBS₁₀, 100 mg/ml Gentamycin). (An additional and even easier way to do this is to resuspend your cells in their regular complete medium and then add the DMSO to 5% of the volume.)
3. Resuspend cells and add 1 ml per freeze vial.
4. Place sealed vials in freezing apparatus and adjust depth of vials properly as demonstrated.
5. Place the freeze unit in a nitrogen freezer that is at least one half full of liquid nitrogen.
6. After 3 hr place the vials on canes and place the canes in the appropriate freezer cans.  (WE WILL DO THIS FOR YOU.)

B. Recovery of Frozen Cells
1. Remove a vial from the nitrogen freezer and rapidly thaw at 37C.
2. Carefully pipette the contents of the vial onto 10ml of prewarmed medium contained in a 10ml centrifuge tube. Centrifuge at 4-600 rpm for 5 minutes. Aspirate off the media carefully so as not to disturb the pelleted cells at the bottom and resuspend the pellet in the quantity of prewarmed medium to be used for the size flask in which the cells will be planted. (5ml for a T-25; 15ml for a T75, etc.)
3. Incubate the cells in a CO₂  incubator. Once the cells have attached to the flask, usually within 4-6 hours but certainly by the next day, one can remove the medium containing possible residual DMSO and add fresh growth medium. Depending upon the cells you are using this may or may not be necessary--examine and cells and decide if they are exhibiting toxic signs.