Skip to content

Passaging Adherent Cells

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to passage adherent cell lines. Unlike suspension cells, adherent cells stick to the bottom of the glassware they are in. Therefore, they are surface area limited rather than volume limited. The most common adherent cells are HEK 293T cells, which are commonly used for virus production. While the procedure for most adherent cell lines are the same, different culture conditions and growth media might be required.

Reagents and Materials

  • Trypsin
  • Growth Media
  • An Existing Cell Culture
  • TC treated or Non-TC treated glassware ^1
  • 50mL Centrifuge tubes
  • PBS

Things to Note before starting

Looking out for contamination

Since these cells exist on the surface of the plate, do not be alarmed if the surface appears "hazy" - that is normal. What you should be concerned is if the media above the bottom becomes cloudy and opaque. If that occurs, bleach the plates and rinse the culture down the sink.

Estimated Timing

This procedure should take slightly longer than passaging suspension cell lines. Approximately 45 minutes to 1 hour. This should be done every 2-3 days (ie Monday / Wednesday / Friday OR Monday / Thursday).

Procedure

  1. Remove the cell culture from the incubator and perform a visual inspection to ensure there is no contamination.
  2. Aspirate off the media on top of the cell culture, making sure not to disturb the cell layer.
  3. Add 10mL of PBS to the plate.
  4. Add 5mL of Trypsin to the plate.
  5. Incubate back in the 37C incubator for 2 minutes.
  6. Using a serological pipette, resuspend the cell layer in the Trypsin / PBS mixture and transfer to a 50mL Centrifuge Tube.
  7. Wash the dish with 10mL of Growth Media and transfer to the 50mL Centrifuge tube to deactivate any excess Trypsin Enzyme.
  8. Spin down the tubes at 800 x g for 5 minutes.
  9. Aspirate off the supernatant and resuspend the pellet in 5mL of Growth Media.
  10. Count cells with the Countess to calculate the cell concentration.
  11. Seed the cells in a new plate with new Growth Media at the appropriate seeding concentration. 2
  12. Return the plates to the 37C incubator.

Next Steps

While passaging the cells, not all the current cells will be needed to create the next passage. Usually, if the passage number is low, these leftover cells are frozen down and stored in liquid nitrogen to use at a later date.

Appendix

Footnotes

  1. The choice to use TC treated or non-TC treated glassware is a somewhat personal choice. Usually you can be okay using them interchangeably. ↩

  2. For HEK 293T Cells, I seed 2e6 cells in 20mL of Growth Media inside of a 150mm dish. ↩