Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
1.? Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min.
2.? Adjust the concentration of cells to 2 x 106 - 2 x 107/ml in fresh medium.
3.? While cells are centrifuging prepare a 5% (v/v) solution of sterile DMSO in fresh medium.
4. Mix the cell preparation and the 5% DMSO in equal portions. Thus, the final concentration will be 106 - 107 cells/ml and 2.5% (v/v) DMSO. The time from the mixing of the cell preparation and methanol stock solution to the beginning of the freezing process should be no less than 5 min and no greater than 15 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.?? Store in either the vapor or liquid phase of a nitrogen refrigerator.
8.?? 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 thawed.
9.?? Immediately after thawing, aseptically transfer contents to a 16 x 125 mm screw-capped test tube containing 7 ml of ATCC Medium 1251.
10.????????? Incubate the tube upright with the cap screwed on loosely (loosened one half turn) at 25°C.
