Recommendations for handling of cryopreserved cells
1. The cell is packaged by dry ice. When receving the cell, please make sure that the vial is still frozen. If there is cell thawing in the tube, Please take photo before expreriment or storage.
2. If immediate culturing is not intended, the cryovials must be stored in liquid nitrogen (-196°C) or at least at -80°C after arrival.
3. Qucikly thaw by rapid agitation in a 37°C water bath within 45-90 seconds. The water bath should have clean water containing an antimicrobial agents. As soon as sample has thawed, remove the cryovial from the water bath.
4.Transfer the cryovial to a sterile flow cabinet and wipe with 70% alcohol. Carefully open the vial and transfer the cell suspension into a 15 ml centrifuge tube containing 9 ml of cell complete medium (room temperature or 37°C ) .
5. In order to reduce cell damage, add 1 ml of cell complete medium into cryovial, slightly pipette, then use a pipettor to add 1 ml of suspension into the centrifuge tube. Resuspend the cells carefully. Centrifuge at 300xg for 3 min and discard the supernatant.
6. Resuspend the cells carefully in 5-10 ml fresh cell culture medium and transfer them into one T25 cell culture flasks or 60-mm diameter dishes.
Cell culture and Passage
1. Determine viable cell count.
2. Seed the dish or flask at the cell density at 1.93-2.94x10^5 cells/ml.
3. Incubate at 37°C in a 95% humidified, 5% CO2 atmosphere incubator.
4. Medium changing and passage by dilution.
5. Freeze cells when not intended for use and store in liquid nitrogen (-196°C).
