(Cell Passaging, Cell Splitting)

This protocol is a Step-by-Step guide to passage adherent cells (e.g. A549, MCF7, HepG2) from a 75 cm² cell culture flask (T75) to a new T75 flask. At the end of the protocol are volumes for other vessel formats.

Materials

All consumables and solutions must be sterile at all times!

• Adherent cells in a 75 cm² cell culture flask (T75) at late log phase
  (cells occupying 80% to 90% of the flask bottom; reaching 90% confluence)
• 1x Phosphate Buffer Saline (PBS); without calcium and magnesium
• Trypsin-EDTA 0.05% Solution
• Complete cell culture medium suitable for the cell line; supplemented with fetal bovine serum (FBS)
• Serological pipettes (5ml, 10ml, 25ml), 50ml conical tubes
• New T75 cell culture flask
• Equipment:
  Laminar flow hood, electronic pipette aid, spray bottle with 70% ethanol,
  water bath at 37°C (optional)
• Optional:
  Automatic or manual cell counter

Preparations

• Wipe down the interior surfaces of the flow hood with 70% ethanol.
• Wear gloves and a lab coat throughout all procedures, and spray your hands with 70% ethanol before working inside the flow hood.
• Allow PBS, Trypsin-EDTA solution, and cell culture medium to warm to room temperature (30–45 minutes). Alternatively, warm PBS and the medium in a 37°C water bath, noting that the water bath can be a potential source of contamination.
• Place all necessary materials under the flow hood and thoroughly spray each item with 70% ethanol before introducing them to the hood.
• Take the T75 flask with cells out of the CO₂ incubator and examine it under the microscope for the following:
       Confluence (cells covering 80% to 90% of the flask)
       Contamination (yellow and/or cloudy medium)
• Place the flask under the flow hood, and spray your hands again with 70% ethanol before beginning work inside the hood.

Procedure

1. Rinsing the cells

Before dissociating the cells, the old medium must be discarded, and any residual FBS from the medium must be washed off. If not removed, the residual FBS will inhibit the trypsin activity, and the acidic pH of the exhausted medium could also interfere with the trypsinization process.

• Open the flask, tilt it slightly, and aspirate the medium using an aspiration pipette connected to a suction line or a serological pipette and dispose of the old medium into a designated waste container.
• Recap the flask and discard the aspirating pipette. Best Practice: Dispose of used pipettes after each step to maintain sterility. Keep all vessels, flasks and bottles closed!
• Add 10 mL of 1x PBS (without Ca/Mg) to the flask (0.2 mL/cm²). Hold the flask at an angle and dispense the PBS along the sidewall to avoid disturbing or dislodging the cells.
• Gently rinse the cells by carefully swirling the flask to distribute the PBS evenly; 2 - 3 times.
• Aspirate the PBS using an aspiration pipette connected to a suction line or a serological pipette, and dispose of the PBS into a designated waste container.

2. Cell Dissociation

• Uncap the flask and add 2 ml Trypsin-EDTA solution directly to the cells.
• Gently swirl the flask to ensure the Trypsin solution evenly covers all the cells, then close the flask.
• Incubate the flask at 37°C for 3–5 minutes (incubation time may vary depending on the cell type). During this time, the cells will round up and detach from the bottom of the flask. After 2 minutes, examine the cell dissociation and rounding under the microscope. When the flask is tilted, the cell monolayer should smoothly detach and slide down the surface.
       - Do not leave the flask in the incubator longer than necessary.
       - However, avoid forcing the cells to detach before they are ready, as this may cause damage or
         clumping.

3. Collecting

• Add 8 mL of complete cell culture medium (with FBS!) to the flask and disperse the cells by gently pipetting over the surface where the cell monolayer was attached (3 to 5 times).

• Pipette the cell suspension up and down several times, keeping the pipette tip at the bottom corner of the tiled flask. Avoid creating foam to prevent cell damage.
      - Pipette the suspension up and down sufficiently to achieve a single-cell suspension.
      - A single-cell suspension is essential for accurate cell counting and even cell distribution in the new
        flask, ensuring uniform attachment and growth.
      - The required pipetting intensity varies between cell lines. Excessive pipetting can cause mechanical
        damage due to shearing forces. Primary cells and early-passage cells are more sensitive to shear
        stress than long-established cancer cell lines, which have been subcultured for decades.

• For routine culturing with a splitting ratio, keep the cell suspension in the upright T75 flask until the new flask is prepared for seeding. For cell counting and dilution, transferring the cell suspension to a 50 mL conical tube is preferable.
      - For well-established standard cell lines such as A549, MCF7, or HepG2, a waiting time of up to
         10 minutes is acceptable.
      - This waiting time should be minimized as much as possible.

4. Re-Seeding

• Label the new flask with the following information:
      - cell name,
      - passage number,
      - date,
      - splitting ratio or cell number.
• For weekly routine culture, splitting ratios commonly used range from 1:5 to 1:20, depending on the specific cell line. Please refer to detailed information in the product sheet.
• For a 1:10 splitting ratio (dilution), add 13.5ml complete cell culture medium to a new T75 flask.
• Resuspend the cell suspension from step 3 thoroughly by pipetting the suspension up and down several times.
• Add 1.5ml cell suspension to the new T75 flask with the medium. In total the flask contains 15ml medium with cells.
• Close the flask and gently swivel the flask using horizontal and vertical rocking motions.
      - Never swivel in circular movements, as this will cause cells to accumulate on the sides of the
         flask, leading to uneven distribution.
      - Even distribution is essential to achieve a proper cell monolayer in the new flask.
• Incubate the flask in a cell incubator at 37°C, 5% CO₂, and 95% humidity.
  
  

Useful numbers for other vessel formats