1. ? Prepare a 20% (v/v) sterile DMSO solution in fresh ATCC Medium 1796.?
2.?? Transfer a culture at peak density to centrifuge tubes and spin at 230 x g for 5 minutes.
3.?? Remove the supernatant and resuspend the cells in ATCC medium 1796 to a concentration of 2 x 106 cells/ml.
4.?? Add the cryoprotectant solution, prepared in step 1, to the cell suspension in three equal aliquots every 2 minutes.? The cell suspension should be at a 1:1 ratio with the cryoprotectant solution when it reaches its final volume.? This will give a solution of 10% DMSO and 106 cells/ml.
5.?? Distribute the cell suspension in 0.5 ml aliquots into 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).? The time from mixing the cell preparation and the DMSO solution, before the cooling cycle begins should be no less than 15 min and no more than 30 min.
6.?? Place the vials in a controlled rate freezing unit.? Use the following cooling cycle: From room temperature cool at
-10°C/min to the heat of fusion; from the heat of fusion to??
-40°C cool at -1°C/min.?? At -40°C plunge into liquid nitrogen.
7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.
8.?? To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the vial.
9.?? Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 5 ml of fresh ATCC medium 1796 in a 16 x 125 mm screw-capped test tube. Incubate upright at 25°C with caps loosened one-half turn.
