1.?? Harvest cells from a culture that is at or near peak density. To detach cells from the wall of the culture tubes place on ice for 10 minutes.? Invert tubes several times until the majority of the cells are in suspension.? Centrifuge tubes at 800 x g for 5 minutes.?
2.?? Adjust the concentration of cells to 2 x 107/ml in fresh medium.
3.?? Before centrifuging prepare a 24% (v/v) solution of sterile DMSO in fresh medium containing 8% (w/v) sucrose.? The solution is prepared as follows:
a) Add 10.5 g sucrose to 10 ml of fresh medium and filter sterilize through a 0.2 mm filter;
b) Add 2.4 ml of DMSO to an ice cold 20 x 150 mm screw-capped test tube;
c) Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 7.6 ml of ice cold medium prepared in step 3a.? The final concentration will be 24% (v/v) DMSO and 8% (w/v) sucrose;
d) Invert several times to dissolve the DMSO;
e) Allow to warm to room temperature.
4.?? Mix the cell preparation and the cryoprotective agent, prepared in step 3, in equal portions. Thus, the final concentration will equal 12% (v/v) DMSO + 4% sucrose (w/v) and 107 cells/ml. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 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.
8.?? To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just to a level just above the surface of the frozen material. Do not agitate the vial.
9.?? Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate a 16 x 125 mm screw-capped test tube containing 13 ml ATCC Medium 2695 cooled to a temperature not above 18°C.
10.????????? Incubate the culture on a 15° horizontal slant at 15-18°C.
