Passaging, also known as subculturing, is a routine procedure in cell culture to maintain healthy cell growth and prevent over-confluence. Here is a general outline of passaging adherent cells:
- Prepare fresh culture medium: Make sure you have enough fresh culture medium appropriate for your specific cell line, warmed to 37°C.
- Inspect the cells: Check the cells under an inverted microscope to ensure they are healthy and have reached the desired confluence for passaging (typically 70-90% for adherent cells). Also, ensure that the cells are free of contamination.
- Aspirate the old medium: Remove the old medium from the cell culture flask or plate using a sterile pipette or vacuum aspirator, being careful not to disturb the cell monolayer.
- Wash the cells: Add an appropriate volume of pre-warmed phosphate-buffered saline (PBS) to the culture vessel to wash the cells and remove any residual medium. Gently swirl the plate or flask to ensure even coverage, then carefully aspirate the PBS.
- Add dissociation agent: Apply an appropriate amount of pre-warmed dissociation agent (e.g., trypsin-EDTA solution) to cover the cell monolayer. Incubate the cells at 37°C for the required time (usually 1-5 minutes, depending on the cell line and the dissociation agent).
- Monitor cell detachment: Observe the cells under the microscope to ensure they have detached from the surface. Gently tap the side of the culture vessel to facilitate detachment, if necessary. Avoid overexposure to the dissociation agent to prevent cell damage.
- Neutralize the dissociation agent: Add an equal volume of fresh, pre-warmed culture medium containing serum (or a trypsin inhibitor) to the culture vessel to neutralize the dissociation agent. Gently mix the medium and dissociation agent to ensure even distribution.
- Collect and resuspend the cells: Transfer the cell suspension to a sterile centrifuge tube, and centrifuge at an appropriate speed (typically 200-300g) for 3-5 minutes to pellet the cells. Aspirate the supernatant carefully, ensuring not to disturb the cell pellet, and resuspend the cells in an appropriate volume of fresh, pre-warmed culture medium.
- Count and seed the cells: Count the cells using a hemocytometer or automated cell counter, and calculate the number of cells required for seeding into new culture vessels. Seed the cells at the recommended density for your cell line, ensuring even distribution across the culture surface.
- Incubate and monitor: Place the newly seeded culture vessels in a humidified incubator at 37°C and 5% CO2. Monitor the cells regularly for confluence, morphology, and contamination.
Always follow the specific guidelines for your cell line and maintain aseptic techniques throughout the passaging process to ensure successful cell growth and to prevent contamination.
