Harvest cells from a culture which is at or near peak density by centrifugation at ~800 x g for 5 min.

Adjust concentration of cells to 2 x 10⁷/mL in fresh medium.  If the concentration is too low, centrifuge at ~800 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.

While cells are centrifuging, prepare a 10% (v/v) solution of sterile DMSO in fresh medium (broth).  The DMSO solution when first prepared will warm up due to chemical heat. The solution should be allowed to return to room temperature prior to use.

Mix the cell preparation and the DMSO solution in equal portions. The final concentration will be 10⁷ cells/mL and 5% (v/v) DMSO. 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 more than 30 min.

Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).

Place the ampules 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.)  If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion.  At -40°C, plunge ampules into liquid nitrogen.

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

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 it is thawed.

Remove the vial from the water bath immediately after thawing.  Aseptically transfer the contents of the ampule into a T-25 tissue culture flask containing 10.0 mL ATCC medium 44 supplemented with 10% HIFBS and 10 μg/mL hemin. 

Incubate the tube at 20-25°C with the cap screwed on tightly.

Maintain as described above.