6.1. Cell Culture Protocol

Cell Thawing and Growth

1. Retrieve a cell vial from liquid nitrogen storage. Keep on dry ice until ready to thaw.
2. When ready to thaw, swirl the vial of frozen cells for approximately 60 seconds in a 37°C water bath. Once cells are thawed (it may be slightly faster or slower than 60 seconds), quickly transfer the entire contents of the vial into a 10 ml pre-warmed RPMI1640 complete medium (with 10% FBS) in a 14 ml conical centrifuge tube.
3. Immediately spin down the cells at 300 x g for 5 minutes, remove the medium, and resuspend the cells in 5 ml of pre-warmed RPMI 1640 medium.
4. Transfer the resuspended cells to a T25 flask and incubate at 37°C in a 5% CO₂ incubator.
5. After 24 hours of culture, check for cell viability. For a T25 flask, add 3-4 ml of RPMI1640 medium, and continue growing in a 5% CO₂ incubator at 37°C until the cells are ready to passage.
6. Cells should be passaged when they reach a density of 2 × 10⁶ cells/ml.

*Note: Leaving the cells in the water bath at 37°C for too long will result in rapid loss of viability.

Cell Freezing

1. Spin down the cells at 300 x g for 5 minutes, remove the medium, and resuspend the cell pellet in 4°C Cell Freezing Medium (FBS at10%DMSO) at a density of ~2 × 10⁶ cells/ml.
2. Dispense 1 ml of cell suspension into each cryogenic vial. Place the vials in an insulated container for slow cooling and store at -80°C overnight.
3. Transfer the vials to liquid nitrogen the next day for long-term storage.
   

*Note: It is recommended to expand the cells and freeze at least 10 vials at an early passage for future use.