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.xa0xa0 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.xa0 Invert tubes several times until the majority of the cells are in suspension.xa0 Centrifuge tubes at 800 x g for 5 minutes.xa0

2.xa0xa0 Adjust the concentration of cells to 2 x 10⁷/ml in fresh medium.

3.xa0xa0 Before centrifuging prepare a 24% (v/v) solution of sterile DMSO in fresh medium containing 8% (w/v) sucrose.xa0 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.xa0 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.xa0xa0 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.xa0xa0 Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

6.xa0xa0 Place the vials in a controlled rate freezing unit.xa0 From room temperature cool at -1°C/min to -40°C.xa0 If the freezing unit can compensate for the heat of fusion, maintain rate atxa0xa0xa0xa0xa0xa0xa0 -1°C/min through the heat of fusion.xa0 At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately

xa0xa0xa0xa0xa0 -1°C/min.) xa0

7.xa0xa0 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.xa0xa0 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.xa0xa0 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.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Incubate the culture on a 15° horizontal slant at 15-18°C.