I am working with neuro2a cell line.I grow them in DMEM with 4.5g/L glucose medium. For the last few months, cells are forming debris and have started looking stressed. This problem persists even if the medium is changed on alternate day. Also, cell growth has been slowed down. The culture has been tested negative for mycoplasma contamination. Around 1 year back, cells of the same passage number were growing very well even in DMEM with 1g/L medium.Can anyone help me in this context? Attaching few images of the same.
The cells look stressed. Do you add FBS to the DMEM? I supplement the high Glucose DMEM with 10%FBS and Glutamax for maintaining the culture. For imaging or staining procedures I usually seed them in DMEM with 1% FBS.
Check the following conditions if FBS is the same:
1. Increase the seeding density. [I split somewhere between 1:3-1:5 for a T-25/T-75 flask and for 12 well plate I seed around >=200000 cells/well.]
2. Reduce time for trypsinization.[I tend to keep it usually for 2-5 minutes.]
or
3. You can try thawing out a fresh batch of cells and try the above suggestions on the fresh batch.
If you want to rule out bacterial contamination (which is also a possibility), just don't add antibiotics to the media while seeding. If it is contaminated, you will see a lawn of bacteria surrounding your cells the next day. In which case you will have to thaw a fresh batch.
Hope it helps.!
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