1.Cell Resuscitation and Culture

1) Preheat the medium in advance, prepare the cell culture dish and centrifuge tube, and add 2ml of preheated medium to the tube.

2) Thawing cells: Take out cells from -80℃ or liquid nitrogen, put them directly into a 37℃ water bath, thaw while shaking, about 5min.

3) After thawing, wipe the cryogenic vial with alcohol and move it into a biological safety cabinet, then add the liquid in the cryogenic vial to the centrifuge tube containing the 9mL culture solution.

4) Centrifuge at 800r for 5min, remove the supernatant after centrifugation, add 2ml of complete medium to resuspend the cells, and transfer cells to a T25 flask containing 4 mL of pre-warmed Cell Culture Medium.

5) Place the cells into the incubator at 37℃ with 5% CO₂. The time of change new medium was determined by cell conditions.

Notes:

--After taking out the frozen cells, put them in a 37℃ water bath to thaw as soon as possible, and the whole recovery process should be fast;

--When putting the cryogenic vial into or taking it out of the liquid nitrogen container, it is necessary to do a good job of protection to avoid frostbite;

--Mix the cells by flicking gently, do not repeatedly pipette.

2. Passaging Adherent Cancer Cell Lines

1) Remove the old culture media, gently wash the cells with PBS. Then aspirate the PBS from the flask.

2) Add digestive enzyme (0.25% Trypsin-0.53mM EDTA). Rock the flask gently to ensure that digestive enzyme covers the surface of the cells.

3) Incubate the flask at 37℃ for 3-5 minutes. Observe the flask under a microscope to check if the cells have detached.

4) Add fresh media to the flask and transfer the liquid to a sterile tube.

5) centrifugal, 300g, 3min

6) Aspirate the supernatant. Resuspend the cell pellet in fresh media and mix well to achieve a single-cell suspension.

7) Transfer the flask to a 37℃, 5% CO2 incubator. Agitate the flask gently in all directions to ensure even distribution of cells throughout the flask.

8) Monitor the cells daily and record their confluency.

3. Passaging Suspension Cancer Cell Lines

1) Gently pipette the cell culture suspension to disperse cells in the dish, then transfer the solution to a centrifuge tube.

2) Repeat steps 5)-8) above.

Fig. 1 Passaging Cancer Cell Lines Flowchart

4.Freezing Cells

1) Prepare freezing medium. To prevent ice crystals from forming in a solution when it is frozen, the solution should contain a cryoprotective agent such as DMSO or glycerol. Put it store at 2-8℃ until use.

2) Resuspend cells in the medium required for their growth and maintenance.

3) Centrifuge at 4℃, 1,000 rpm, 5min. Remove the supernatant with an aspirator.

4) Resuspend the cell pellet in freezing medium. The cell density is 5*10^5-1*10^7 cells/mL.

5) Transfer the cell suspension into cryopreservation tubes.

6) Store the tubes at -80℃.