I have begun regrowing RAW 264.7 cells from our cryo stock in DMEM with 10% FBS and 1% antibiotics. The cells are very round and much smaller (less than 10 um) than what I have grown in the past. They are only on the 10th (or so) passage. In the past, these cells were 30 um or so in size and were more triangular/stretched in shape. Any help would be appreciated!
Hello Anna,
There can be two possible reasons for this:
1. Your stock is too old and cells are not as good as they used to be, in this case just change media normally and do trypsinization after confluence, cells will achieve normal morphology after 2nd-3rd passage (this type of problem encounters with many cells like HepG2).
2. You have put some cold media or PBS in cells due to which they have become rounded, in this case, revive a fresh stock and take care to add warm (37degree) media.
Hope it helps !
Hi Anna,
Agree with Ajay: take care to add warm (37degree) media after thawing fresh stock. Try seeding the cells in a smaller flask at first and change the medium frequently (every 2 days) at the beginning to remove any dead cells from the supernatant. You can also coat your flask with 5% FCS before seeding the cells as that helps with more fragile cells at the beginning of culture,
Good Luck!
Dear Anna,
Usually, Raw264.7 are round with no spread over the cell surface. They start to be sharp when they are too confluent or activated by differentiation factors.
Regards
Yes growing cells can be a tricky business. The reason why your cells are so tiny could be they're getting ready for apoptosis, and this should be expected if your just removing them from liquid nitrogen. Not all cells will survive the freezing process. Also some cells can be pretty slow growers and take weeks before they start looking and dividing normally. As previously mentioned how you culture the cells is very important, you might want to stick to how your group cultures those cells, just to maintain consistency. But i would say be wary of how you split the cells, It could be your splitting at a higher ratio. Just a few things to keep in mind when doing tissue culture work. Good luck with your cells.
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