1.?? Two to three days in advance, prepare a 14% (v/v) sterile glycerol plus 14% (v/v) sterile DMSO solution in Stone's Modification of Locke's Solution in the following manner:
????? a) Combine 0.84 ml of sterile glycerol and 0.84 ml of sterile DMSO in a 16 x 125 mm screw-capped test tube.? Chemical heat will be liberated from this combination so allow the solution to cool to room temperature.
????? b) To the glycerol/DMSO solution add 4.32 ml of Stone's Modification of Locke's Solution.? Mix by inverting several times.
????? c) Loosen the cap one full turn and place in an anaerobic jar with an anaerobic GasPak for 2-3 days.
2.?? When the test tube cultures are at or near peak density remove the tubes from the anaerobic jar and immediately screw the caps on tightly. One by one gently remove the cells from the bottom of the egg medium slants and pool in a single 16 x 125 mm screw-capped test tube (work quickly to avoid prolonged exposure to air).?
3.?? Adjust the concentration to 1.0-2.0 x 107cells/ml using overlay from a reduced tube of medium.? If the concentration of cells is too low centrifuge at 500 X g for 5 minutes.? Adjust the volume of supernatant to yield the desired final cell concentration.
4.?? Mix the cell preparation and the cryoprotective agent, prepared in step 1, in equal portions. Thus, the final concentration will equal 7% (v/v) glycerol, 7% (v/v) DMSO and 5.0 x 106 - 1.0 x 107 cells/ml. 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 longer than 30 min.
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.? 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 1.5 to 2 hours and then plunge ampules into liquid nitrogen.? (The cooling rate in this apparatus is approximately
????? -1°C/min.) ?
7.? The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated.
8. ? Before thawing an ampule do the following.? Loosen caps one full turn and place tubes containing ATCC medium 1671 and 25% HIHS in an anaerobic jar.? Add a BBL GasPak (one anaerobic system GasPak per BBL GasPak 100 anaerobic culture jar).? Close the vessel securely and incubate at 35°C for at least 48 hours.? The palladium catalyst in the GasPak jar should be replaced biweekly with fresh catalyst.
9.?? Thaw the frozen ampule in a 35°C water bath without agitation until all of the contents are liquid (about 2-3 minutes).
10.????????? Aseptically and gently, lower a sterile Pasteur pipette from which the air has been expelled to the bottom of the liquid in the ampule and slowly aspirate the entire contents into the pipette.? Be careful to minimize agitation of the fluid and so not introduce air bubbles from the tip of the pipette.
11.????????? With the cap of the test tube loosened one full turn place it in an anaerobic jar containing a BBL GasPak and incubate at 25°C for at least 48 hours.
12.????????? Subculture every 2-3 days.
