Hello,
I have been cultivating RAW264.7 cells from ATCC recently. I culture them in DMEM medium + 10% FBS at a density of 10,000 cells/cm² in a T75 flask with a volume of 10mL.
For cell passages, I use trypsin to detach the cells.
During the first two days of culture, they reached 60% confluence after 15 hours, and I performed a cell passage. Then, after the second passage, it took them 8 days to reach confluence. I noticed a change in morphology on the third day after the second passage, with an unusual morphology of cluster formation. (Attached photos)
Could someone help me understand why these cells behave like this and why they take so long to grow?
Thank you in advance,
Hello Melisa Ait Kaci
The cells are not looking good. RAW 264.7 cells are semi-adherent i.e., some cells grow in suspension, some loosely attach to the surface and others flatten out and attach to the flask. Cells should not be allowed to overgrow and become confluent as this can lead to flattened adherent cell characteristics. Allow the cells to become about 60-70% confluent.
You may incorporate these measures in your protocol.
1. RAW 264.7 cells are never to be trypsinized. Dislodge cells from the flask substrate with a cell scraper; aspirate and add appropriate aliquots of the cell suspension into the new culture vessel. Lifting these cells can be tricky sometimes, in which case, accutase or lidocaine in PBS may be used.
2. A sub-cultivation ratio of 1:3 to 1:6 is recommended. The greater the dilution, the longer the cells will take to reach confluency. So, do not heavily dilute as cells seeded with extremely low confluence will exhibit impaired growth and proliferation. You may increase the cell density in a T-75 cm^2 flask. Instead of 10,000 cells/cm^2 make it to 40,000 – 50,000 cells/cm^2 with a volume of 15ml.
3. The growth media should be refreshed every 2-3 days but no more than 4 days without causing significant cell death.
4. What is the passage number? Do not use cells above passage number 20 because cells start growing slowly after passage 15-20.
Best.
Hello Malcolm Nobre ,
Thank you so much for your detailed reply.
Can you enlighten me as to why trypsin should not be used? It's true that sometimes cells have trouble being detached even after 20min of tryspsination.
As for the number of passages, I'm at P6, at the moment. But on the photos, they were at P4. For these cells, I'll stop at P12.
Best regards,
Melisa
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