1.?? Harvest the culture by agitating the contents of each flask.? Transfer the cell suspensions to 15 ml plastic centrifuge tubes.

2.?? Spin the cell suspensions at approximately 500 x g for 5 min.

3.?? Pool the cell pellets and adjust the concentration to 2.0 - 4.0 x 10⁷ cells/ml with a fresh ATCC medium 1728.

????? *If the concentration is too low centrifuge at 500 x g for 5 min and resuspend in the volume of ATCC medium 1728 required to yield the desired concentration.

4.?? Mix the cell preparation and 20% (v/v) DMSO in equal portions.? The final concentration will be 1.0 - 2.0 x 10⁷ cells/ml and 10% DMSO.? The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.

5.?? Dispense in 0.5 ml aliquots to 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 2.5 to 3 hours and then plunge ampules into liquid nitrogen.? (The cooling rate in this apparatus is approximately

-1°C/min.) ?

7.?? Store in either the vapor or liquid phase of a nitrogen refrigerator.

8.?? To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.? Do not agitate the ampule.? Do not leave ampule in water bath after thawed.

9.?? Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing 10 ml ATCC medium 1728.

10.????????? Incubate in a 25°C CO₂ incubator with the caps screwed on tightly.