We are trying to differentiate primary human monocytes from 30 ml of peripheral blood into monocyte-derived macrophages for differentiation studies.
However, we are losing cell viability quite drastically and end up with not enough cells to run experiments.
Following PBMC isolation - using Ficoll and negative selection with Miltenyi monocyte isolation kit II - we allow PBMCs to adhere to TC-treated 24-well plates (8x105 per well) containing 1 ml of RPMI-1640 supplemented with pen/strep 1% (v/v), 10% FBS and M-CSF (20ng/ml).
After a media change on day 3, we harvest cells at day 7 using Accutase. Although PBMCs adhere on the day of isolation and look beautiful on the first few days, they are often loosely-adherent and loose viability after day 4.
We typically obtain 3,5x107 PBMCs after isolation, 4,5x106 cells following magnetic separation and harvest 8x105 monocyte-derived macrophages at day 7. Most of these cells collected at day 7 are already dead (cellular yield around 18%). I would have to further differentiate these cells into M1, M2 at this point, but so far I have been unable to obtain a reasonable cell count.
Any suggestions on how to avoid these problems? Maybe the concentration of M-CSF, non TC plates, humam serum?
Shall I use tissue culture treated polystyrene plates or non-tissue culture treated ones? Any particular recommendation?
Does it make a difference using human serum from AB clotted whole blood or AB plasma?
Around 10% of cells among PBMCs are supposed to be monocytes and so your yield isn't the problem. Do you know whether your monocytes are stressed after MACS isolation? You could try gentler isolation methods that don't involve the use of columns. I think the problem is that you are putting a less than optimum number of monocytes/well during your culture. Try putting 2-3x10e5 monocytes/well. You could also increase the human M-CSF dose and do media changes on D2, D4 and then harvest the cells on D7.
I woulnd't add human serum since it might contain proteins (esp. cytokines) that could have an impact on your polarization studies.
I've never tried using M-CSF... usually GM-CSF works better. I can't remember the concentration I used to use, but I think 20 or 40 ng/mL should be more than enough.
Your cell concentration is probably the problem. Cells do not like "space". I would not go below 0.5x10e6 cells/well. I usually use cell concentration at 1x10e6 cells/mL and plate 0.5mL/well in 24 well plates.
Second, I would NOT try to "re-isolate" the macrophages once they are stuck to the plate. Just differentiate them in the wells.
Heat inactivated FBS is fine, you don't need human serum.
Always use cell culture treated plates, that's not the problem. And I'm pretty sure all cell culture plates are polystyrene (the hard, clear plastic, not polyproplyene, which is what we usually use for 15 and 50mL tubes since they can spin at higher rpm.)
You don't need to increase your media change rate. Once at D3, and maybe once at D5 is enough. Make sure that you remove the dead cells during your media change, otherwise, they tend to accelerate more cell death.
If you need more cells, I would highly suggest you increase the amount of blood you are taking.
Just as a follow up to Shen-An Hwang's comment:
I assumed Mariane's protocol went like this- isolate PBMCs and then isolate monoytes using Miltenyi kit, then let the monocytes adhere to plates, media change on D3 and harvest on D7.
GM-CSF would give a mix of monocyte derived dendritic cells and macrophages. M-CSF is recommended if you want a purer population of macrophages. We do two rounds of media change before harvest on D7 and have gotten good yields. We do D2 and D4 but I agree that D3 and D5 should work the same.
Coat the plates you are using to culture them with human fibronectin (20 ug/ml), and optimize the M-CSF or GM-CSF concentrations that you are using (don't assume that they are 1005 active and dont freeze thaw the stick solutions). Don't plate the monocytes too sparsely.
We use Costar non treated tissue culture plates (#3738) and find that monocytes stick in much greater numbers to them compared to TC-treated plates. We also use human serum (Sigma H4522) from AB plasma to differentiate the cells but I don't know whether this will affect polarization as was commented on in one of the other answers. Detaching macrophages from the wells is always difficult to do and reduces viability quite a bit so we always try to differentiate the macrophages in the plates in which the experiments will be carried out, if at all possible.
Hi! I am trying to differentiate human monocyte to macrophages using M-CSF but I am having some problems! There are lots of papers but much of them indicates different concentration of M-CSF.
Which is the best concentration? I Want to polarize the macrophages to M1 and M2 after differentiation...When I did the polarization (48 hs LPS 100 ng/ml IFNy 20 ng/ml ; IL4/IL13 20 ng/ml respectively) after 7 day with 50 ng/ml M-CSF all of them are M2 (up regulation of CD206 by FACs)
Can you help me?
Thaks!!!
Florencia Veigas Probably a bit late jeje, but hopefully someone finds this helpful. For M-CSF concentration, if you are using human serum (10%) you can use 5-10 ng/ml. If you are using FBS (10%) I will recommend using at least 20ng/ml. For polarizing macrophages 24hrs is enough time. All the other concentrations look fine, you are probably using LPS at its maximum concentration in huMDMs.
Mary P. O'Sullivan How did you go about imaging the differentiated macrophages? I'm having problems lifting mine with good viability but really need to do confocal for my experiment. Are there special glass wells I can culture and differentiate them in for 2 weeks?(I'm using earlier progenitors that need 2 weeks of differentiation)
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