When should I subculture my cells?

Two general criteria can be used to determine the need for subculture: cell density and exhaustion of medium.

• Cell density: Both adherent and suspension cultures should be passaged when they are in the log phase, before reaching confluency. For adherent cells, they stop growing after reaching confluency due to contact inhibition, and it will take them longer to recover when reseeded. On the other hand, cells in suspension will clump together once they reach confluency, and the medium will appear turbid when the culture flask is swirled.
• Exhaustion of medium: Cells should be passaged when a rapid drop in pH (>0.1-0.2 pH unit) is observed, indicating exhaustion of medium. The drop of pH is caused by the accumulation of lactic acid, a by-product of cellular metabolism, which is toxic to the cells and can affect cell growth. The rate of pH change is generally dependent on the cell concentration. A fast drop in pH indicates a high cell concentration, which exhausts medium rapidly.