Hello everyone! :) I am trying to do macrophage differentiation from mouse bone marrow. I am using RPMI-1640 medium with HEPES and 2-ME and for differentiation 20ng/mL recombinant M-CSF. On the 4th day after isolation, I am renewing the M-CSF by replacing the medium. However, at this point, my cells are starting to die and the remaining ones accumulate in the middle as a straight line. I also tried to change the half of the media while keeping the M-CSF concentration stable but it barely made a difference. I am growing them in T25 flasks. Instead of changing the medium should I just supply new M-CSF with an additional medium?
Besides, on day 7 I am stopping M-CSF treatment. Incubating my cells in M-CSF free medium for 24 hours and after that, I am doing M1 and M2 polarization, but during the untreated 24 hours, my cells also tend to die. Can I treat them for polarization right after differentiation? Or how do you do it?
Any help is appreciated! Thank you!
Get rid of the 2-Me. I'm assuming that you have bicarbonate and 10% FBS in there? what is you seeding concentration? Don't go too far below 1x10e6 cells/mL. And what's the volume you are putting into the T25 flasks? I would stick to around 5mL or so...
Bone marrow cells are easy to differentiate, and they shouldn't start to die around day 4. Yes, there will be dead cells floating, which is why it is always a good idea to change the medium completely. Leaving dead cells in your culture causes more cells to die.
By day 7, resting them in M-CSF free medium should NOT cause any sigificant cells to die to the extend that you should notice it.
Shen-An is correct in that you should get rid of 2-Me, it serves no purpose. Also RPMI is already buffered so HEPES likely wont do much.
We tend never to actually replace the media in the first few days, but just top up existing media, there is probably something made by the macrophages themselves that helps with survival (maybe GM-CSF or more M-CSF, or the macrophages like eating the dead neutrophils, its unclear).
We usually seed 30 million bone marrow cells in 30ml media in a T175 on day 0 (20ng/ml MCSF) so this equates to 4.2 million in a T25 in 4.2ml (pretty much what Shen-An uses). Feeding 10ml on day 3 and 6 works the best and by day 7 you will have lots of macrophages to treat with whatever you want. There will be 50% cell death by day 3 (all the neutrophils and other precursors will die - this is normal and the straight line of dead cells you are seeing, I hypothesise that the new macrophages start eating these dead cells), but if you give lots of media on the day 3 feed they will start to proliferate. On day 7 you will have 15 million macrophages. To much M-CSF feeding triggers more proliferation but may leave the cells slightly 'exhausted'. You may have to experiment with concentrations based on the M-CSF batch also.
M-CSF is a vital growth and survival factor (known for >25 years), its actually required to maintain mitochondrial function. Taking M-CSF away will immediately reduce mitochondrial function and eventually trigger apoptosis (unless cells are so dense they make their own M-CSF, also its possible LPS stimulates autocrine growth factor signalling). IL-4 also acts as a 'growth factor' and can work independently of M-CSF.
If you rest the macrophages with no LPS, no IL-4 and no M-CSF they will begin to die, I am not surprised at all you see this. Everyone can tell you if you go away on the weekend and forget to feed your macrophages M-CSF they will die. Maybe by 16 hours you wont see too much evidence of dead cells, but push that to 36 hours and every cell will be dead. It's possible in Shen-An's culture conditions have sufficient autocrine signalling to allow survival, lab variation with this is common and things get really crazy when people use L929 media.
But really there is no reason to remove M-CSF. I cannot think of a time in the body of a human or mouse when you have a infection and all the M-CSF (or IL-34) that is present in the tissue (at pretty decent concentrations - a steady 10ng/ml in the peritoneum) magically disappears to allow a 'pure' LPS or IL-4 signal. These things work in concert, keep it in, keep the cells happy. If your results are different with M-CSF then its possible this is more physiologically relevant, and I would be happy to have a discussion with anyone who disagrees. (This also brings up the possibility of combining GM-CSF and M-CSF, but that's another issue)
Hope this helps,
Best of Luck,
Luke
Thanks so much for the detailed and clear answers. My seeding concentration is 2x10^6 cells/ml and I am seeding 8 million cells to T25 flask (4ml). In the end I did not replace the medium. Instead I fed 2ml medium on day 3 and 4 ml more on day 6. My cells were happy and actually a lot more confluent than ever. Then, instead of removing M-CSF completely, I decreased it from 20ng/ml to 10ng/ml. Today was the last day of my treatments, so hopefully they are successfully polarized into M1 and M2 macrophages and everything is fine. Actually what I saw in the plates were really promising and better than ever so we will see!