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 - 2 x 107/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 - 107 cells/ml and 11% (v/v) DMSO. The time from the mixing of the cell preparation and methanol stock solution to the beginning of the freezing process should be no less than 5 min and no greater than 15 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.? From room temperature cool at -1°C/min to -40°C.? If the freezing unit can compensate for the heat of fusion, maintain rate at??????? -1°C/min through the heat of fusion.? At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.? Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.? (The cooling rate in this apparatus is approximately
????? -1°C/min.) ?
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.? Do not agitate the ampule.? Do not leave ampule in water bath after thawed.
9.?? Immediately after thawing, aseptically transfer contents to a 16 x 125 mm screw-capped test tube containing 5 ml of ATCC Medium 1651.
10.????????? Incubate the tube on a 15° horizontal slant with the cap screwed on loosely (loosened one half turn) at 25°C.
