1. Harvest cells from a culture at or near peak growth by centrifugation at 300 x g for 2 min.
2. Adjust concentration of cells to 2 x 106/ml in fresh
3. While cells are centrifuging, prepare a 13% (v/v) solution of sterile DMSO in fresh medium.
a) Add 1.3 ml of DMSO to an ice cold 20 x 150 mm screw-capped test tube;
b) Place the tube on ice and allow the DMSO to solidify (~5 min) and then add 8.7 ml of ice cold medium;
c) Invert several times to dissolve the DMSO;
d) Allow to warm to room temperature.
4. Add a volume of the DMSO solution equal to the cell
suspension volume but add in 3 equal aliquots at 2 min
intervals. Thus, the final concentration of the preparation
will equal 6.5% (v/v) DMSO and 106 cells /ml.
5. Dispense in 0.5 ml aliquots into 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. Use the following cooling cycle: From room temperature cool at
-10°C/min to the heat of fusion; from the heat of fusion to
-40°C, cool at -1°C/min. At -40°C plunge into liquid nitrogen. The cooling cycle should be initiated no less than 15 and no more than 30 minutes after the addition of DMSO to the cell preparation.
7. Store in the vapor or liquid phase of a nitrogen
8. To establish a culture from the frozen state, aseptically add 0.5 ml of sterile modified PYNFH medium (ATCC Medium 1034) containing 8% (w/v) sucrose to the frozen ampule. Immediately, place in a 35°C water bath, until thawed. Immerse the ampule just sufficient to cover the frozen material. Do not agitate the ampule.
9. Immediately after thawing, aseptically remove the contents of the ampule and gently add the material to the edge of a 20 x 100 mm petri plate containing ATCC Medium 919 (non-nutrient agar) and position on a 15 degree slant. The cell suspension will pool at the edge of the plate.
10.Continue to double the volume of the cell suspension at 10 minute intervals by adding ATCC medium 1034 containing 4% sucrose (w/v). When the volume reaches 16.0 ml place the plate in a horizontal position and incubate at 25°C.
11. On the following day, gently remove the cell suspension for the plate and transfer to a T-25 tissue culture flask. Note the volume of the suspension and add a volume of fresh medium without sucrose equal to the volume of the cell suspension. Incubate the culture at 25°C.
12. After culture has been established subculture into fresh
medium without sucrose.