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Passaging Suspension Cells

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to passage Suspension cells. Suspension cells are cells that grow in a liquid solution, such as Jurkat cells, Raji cells, ML-1 cells, and Stem Cells. "Passaging" means to replace / add more growth media to the suspension culture to ensure the cells have enough resources to continue growing and to ensure that the cells are at an optimum concentration. While each of these cells use different media, the procedure to passage them is similar.

Reagents and Materials

  • Current Suspension Culture of Cells
  • Growth Media
  • TC treated or non-TC treated flasks. ^1
  • 50mL centrifuge tube

Things to Note before starting

Note for specific instructions

Please see RM034 - List of Commonly Used Lab Buffers for a full list of the specific Growth Media. Further information on specific seeding density and whether to use TC treated or non-TC treated glassware can be found online.

Looking out for a contamination

Always be on the lookout for a contaminated culture. A normal healthy culture should be translucent (able to be seen through). If it is contaminated, the culture media will appear cloudy. When looking at the cells under the microscope, if very small dots or black lines are present, that is a sign of a contamination. If the cells are contaminated, bleach the culture and rinse it down the sink

Estimated Timing

This procedure takes 30min - 1 hour and should be done every 2-3 days. (ie Monday, Wednesday, Friday OR Monday and Thursday).

Procedure

  1. Take the current cell culture out of the incubator and perform a visual inspection to ensure the cells are not contaminated.
  2. Count the cells in culture using the Countess to determine the cell concentration. Confirm that the cells have grown since you last passaged them, as lack of growth could indicate something wrong with the conditions.
  3. Pour the cell culture into 50mL Centrifuge tubes. Spin at 800 x g for 5 minutes to pellet the cells.
  4. Aspirate off the supernatant and resuspend the pellet in 1-10mL of media. ^2
  5. Count the cells using the Countess to determine the cell concentration.
  6. Seed the cells in a fresh flask with new growth media at the appropriate seeding density for the cells. 3
  7. Return the new flasks 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. General rule is that suspension cells are culture in non-TC treated cells, but exceptions occur. Ask other members of the lab for their recommendation. ↩

  2. Use the initial cell concentration you got before spinning the culture down to be an estimate of how much media to add. The more concentrated, the more media you should resuspend the pellet in. ↩

  3. The initial seeding density depends on the cell type. For most cells, the seeding density ranges from 0.5e6 - 1e6 / mL. For specific numbers, consult documentation online. ↩