1. Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min.
2. Adjust the concentration of cells to 2 x 10⁶ /mL in fresh medium.
3. While cells are centrifuging prepare a 22% (v/v) solution of sterile DMSO in fresh medium.
4. Mix the cell preparation and the 22% DMSO in equal portions. Thus, the final concentration will be 10⁶ cells/mL and 11% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the beginning of the freezing process should be no less than 15 min and no greater than 60 min.
5. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion.xa0 At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.) xa0
7. The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.
8. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.xa0 Do not agitate the ampule.xa0 Do not leave ampule in water bath after thawed.
9. Immediately after thawing, do not leave in the water bath, gently remove the contents of the ampule with a Pasteur pipette and expel slowly into a 16 x 125 mm screw-capped test tube or T-25 tissue culture flask. xa0Incubate at room temperature (approx. 25°C) for 15 min.
10. At 15 min intervals add 0.25 mL of ATCC medium 1651 dropwise. Continue until the final volume is 2.0 mL.
11. Allow the culture to remain undisturbed for 15 min.
12. Add 0.5 mL of medium dropwise at 15 min intervals until the volume is 4.0 mL.
13. Allow the culture to remain undisturbed overnight.
14. On the morning of day 2 slowly add 4.0 mL of ATCC medium 1651. Allow the culture to remain undisturbed overnight.
15. When the culture is established, follow the protocol for maintenance of culture.

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