Changing Cell Media (General)


This protocol will walk you through the process of changing cell media, also known as feeding cells. Please note that different cell types need to be fed at different frequencies, to be checked on manufacturer protocols, in order to maintain cell culture health.


Methods for Changing Cell Media

  1. Warm cell culture medium and HBSS to 37˚C in the water/bead bath;
  2. Spray them down with ethanol and place them inside a biosafety cabinet (BSC);
  3. Spray down incubator doors with ethanol and minimize breathing to diminish contamination risk;
  4. Remove cell culture flasks from the incubator and place them under a microscope to ensure there is no contamination. See here for more information on bacterial contamination and how to identify it;
  5. Still, under the microscope, check for flask confluency by analyzing the ratio of surface area occupied by cells and surface area not occupied by cells. If confluency is ~80%, follow this protocol on cell passaging;
  6. Spray down flasks with ethanol, wipe them with kimwipes and place them in BSC;
  7. Aspirate cell media from culture flasks, ensuring to avoid touching the bottom of the flask;
  8. Wash flask with HBSS, using the amount needed to cover your cell culture flask surface;
  9. Aspirate HBSS;
  10.  Add fresh cell medium to your flasks;
    • Note: an appropriate volume is usually 0.2 mL/cm2;
  11.  Place your flasks back into the incubator;
  12.  Feeding is usually done every 1-3 days, depending on cell type.

We hope this guide to changing cell media was helpful! Keep in mind that some cell-lines can be trickier than others and don’t always respond to the same technique. Check out our protocol for changing cell media for HUVECs here, or visit the protocols page to learn more cell culture techniques. Happy bioprinting!

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