Tissue Culture Series #3: Cell Passaging

Learn about the sigmoid growth curve of cultured cells, identifying lag phase, logarithmic growth phase, and stationary phase. Understand that cells should be passaged during late log phase at approximately 80% confluency.

Pre-warm culture media and dissociation reagent (trypsin) to 37°C. Verify cells are healthy and contamination-free before proceeding with passaging.

Place flask in biosafety hood, remove old media, and gently wash the cell monolayer with room temperature PBS or HBSS without calcium and magnesium to prepare cells for dissociation.

Add pre-warmed trypsin dissociation reagent to cover cells, tilt flask gently to distribute evenly, and incubate at 37°C for 2-10 minutes. Monitor under microscope frequently until greater than 90% of cells detach.

Add serum-containing medium equal to trypsin volume to neutralize the dissociation reagent. Gently pipette up and down to break cellular clumps and achieve a single cell suspension.

Transfer cell suspension to a conical tube and centrifuge at 300 G for 3 minutes. Discard supernatant and gently resuspend the cell pellet in suitable volume for counting or seeding.

Count cells using hemocytometer or automated counter and transfer the required number of cells to a new pre-labeled flask with pre-warmed medium. Record passage number and initial seeding density for future reference.

For established cell lines with known growth rates, use a predetermined split ratio (typically 1:3 to 1:8) instead of cell counting to transfer a fraction of the cell suspension to new flasks.