Tech Tips for Primary Cell Culture: Common Errors

There is no worse feeling than going into the lab to check your cells only to find your culture is not growing or worse yet not even attached to the flask.

Cell culture can involve a huge cost both financially and in time. No one wants to waste time and money, especially if you’re on a tight budget.
I have compiled a list below of some common errors that should be avoided to keep your primary cells healthy.

Centrifuging the Cells out of Cryopreservation

• For many cell types, the damage to primary cells by centrifugation is much harsher than residual DMSO in the culture media.
• Following the recommended seeding density and recommended volume of media per culture vessel will sufficiently dilute the residual DMSO.
• Changing media the day after seeding cells will remove any residual DMSO.
• Check the Lonza optimized protocol to determine the appropriate thawing and seeding procedure.

Using Alternate Media for Culturing the Cells

• For many of our cell types, Lonza has specialty media specifically formulated for optimal culture conditions.
• Cells can become conditioned to specific culture media. Changing the nutrients can lead to a lack of cell attachment when plating and/or slow proliferation.
  • In particular, endothelial cells can become dependent on VEGF. Therefore, trying to switch to a VEGF-free media can cause the cells to not adhere or grow very slowly.
• Check the Lonza optimized protocol to determine the appropriate media for our cells.

Re-Frozen and Late Passage Cells

• Primary cells are finite and will senescence when passaged too many times.
  • Cells should be used within the specified guaranteed number of doublings
• Some cell types may be able to be re-frozen using our protocol that can be downloaded here, but later passage cells may not be able to be recovered from cryopreservation. In addition, Lonza offers no guarantee for any cryopreserved cells.

Subculturing Reagents

• Primary cells often require lower concentration Trypsin/EDTA formulations for optimal proliferation after passaging.
  • Using too harsh a trypsin can lead to cell death or slow proliferation after passaging.
  • Check the Lonza optimized protocol for the appropriate subculture reagents.

Some of Lonza’s cell culture media are serum free and not effective at neutralizing trypsin activity. Using these media to neutralize trypsin can lead to issues post subculture. Always be sure to use the appropriate neutralizing solution.