Temperature: 20°C to 25°C
DMSO, 1.0 mL
Fresh complete growth medium, 9.0 mL
Harvest and Preservation
- Harvest cells from a culture which is at or near peak density by centrifugation at 800-1000 x g for 5 min.
- Adjust concentration of cells to between 2 x 107 and 2 x 108 cells/mL in fresh medium. If the cell concentration is too low, centrifuge at 800-1000 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 – 108 cells/mL and 5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the start of the freezing process 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 establish a culture from the frozen state, place an ampule in a 35°C water bath (2-3 min). Immerse the vial just sufficiently to cover the frozen material. Do not agitate the vial.
- Remove the vial from the water bath immediately after thawing. Aseptically transfer the entire contents of the ampule into a T-25 tissue culture flask containing 10.0 mL complete medium. Incubate with the cap tightly sealed at 20-25°C.
- Maintain as described above.