Cell Culture and Passaging

Cell Growth

• Follows a standard pattern: lag phase, then exponential (log) phase.
• Passage cells when they cover the plate or exceed medium capacity.
• Maintain log phase for healthy cells.

Preparation

• Sterilize hood and gather supplies before retrieving flasks.
• Examine cultures for contamination/deterioration.
• Handle cells carefully during transport.

Adherent Cell Passaging

• Remove spent medium using a sterile pipette.
• Rinse cells with balanced salt solution (e.g., DPBS) without calcium and magnesium.
• Aspirate the salt solution after rinsing.
• Add cell dissociation reagent (e.g., Triple Express) to cover the cell sheet.
• Tap gently to help cells detach; confirm detachment with a microscope (cells appear round and move).
• Do not leave dissociation reagent on too long.
• Break up clumps by pipetting warmed medium.
• A single-cell suspension is crucial for accurate cell counting.
• Inactivate trypsin with serum or a trypsin inhibitor; Triple Express inactivates by dilution alone.
• Centrifuge cells to remove residual dissociation reagent.
• Remove medium from the centrifuge tube.
• Resuspend the pellet with warm, complete growth medium.
• Remove a sample for counting.

Cell Counting

• Trypan blue indicates live/dead cell ratio (dead cells turn blue).
• Count total and dead cells using a microscope.
• Determine fresh medium addition for optimal seeding density.
• Add required medium, mix gently, and pipette into fresh flasks.
• Cap flasks tightly if vented, loosely if not.
• Distribute cells evenly in the incubator.
• Follow the same counting procedure for suspension cells.

Final Steps

• Add fresh medium to flasks, staying within volume limits.
• Split culture into multiple flasks if needed.
• Cap flasks appropriately and return to the incubator.
• Replace the medium every three weeks to eliminate cell debris and metabolic waste.