1. ? Prepare a 10% (v/v) sterile DMSO solution in fresh ATCC Medium 355.?

2.?? Transfer a culture at peak density to centrifuge tubes and centrifuge at 525 x g for 5 minutes.

3.?? Remove the supernatant and resuspend the cells in ATCC medium 355 to a concentration of 2 x 10⁶ to 2 x 10⁷ cells/ml.

4.?? Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be between 10⁶ and 10⁷ cells/ml and 5% (v/v) DMSO.

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 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.

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 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 355 in a 16 x 125 mm screw-capped test tube. Incubate upright at 25°C with caps screwed on tightly.