Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

1. xa0 Prepare a 20% (v/v) sterile DMSO solution in fresh ATCC Medium 1796.xa0

2.xa0xa0 Transfer a culture at peak density to centrifuge tubes and spin at 230 x g for 5 minutes.

3.xa0xa0 Remove the supernatant and resuspend the cells in ATCC medium 1796 to a concentration of 2 x 10⁶ cells/ml.

4.xa0xa0 Add the cryoprotectant solution, prepared in step 1, to the cell suspension in three equal aliquots every 2 minutes.xa0 The cell suspension should be at a 1:1 ratio with the cryoprotectant solution when it reaches its final volume.xa0 This will give a solution of 10% DMSO and 10⁶ cells/ml.

5.xa0xa0 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).xa0 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.xa0xa0 Place the vials in a controlled rate freezing unit.xa0 Use the following cooling cycle: From room temperature cool at

-10°C/min to the heat of fusion; from the heat of fusion toxa0xa0

-40°C cool at -1°C/min.xa0xa0 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.xa0xa0 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.xa0xa0 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.