1. Prepare a 20% (v/v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath.xa0 Allow the DMSO to solidify.xa0 Add the required volume of refrigerated medium.xa0 Dissolve the DMSO by inverting the tube several times.
   *NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
2. Harvest the culture by agitating the contents of each flask (or by scraping the bottom of each flask using a sterile cell scraper).xa0 Transfer the cell suspensions to 15 mL plastic centrifuge tubes.
3. Spin the cell suspensions at approximately 800 x g for 5 min.
4. Pool the cell pellets and adjust the concentration to 2.0 - 4.0 x 10⁷ cells/mL with a fresh ATCC medium 1728.
   NOTE: 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.
5. Mix the cell preparation and 20% (v/v) DMSO in equal portions.xa0 The final concentration will be 1.0 - 2.0 x 10⁷ cells/mL and 10% DMSO.xa0 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.
6. Dispense in 0.5 mL aliquots to 1.0-2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. 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 ampules into liquid nitrogen. xa0Alternatively, place the vials in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 2 to 3 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.) xa0
8. Store in either the vapor or liquid phase of a nitrogen refrigerator.
9. 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.xa0 Do not agitate the ampule.xa0 Do not leave ampule in water bath after thawed.
10. Immediately after thawing, aseptically transfer entire contents to a T-25 tissue culture flask containing 10 mL ATCC medium 1728.xa0 Incubate with the cap tightly sealed at 20-25°C.
11. Follow the protocol for maintenance of culture.

Thomas A Nerad, personal communication