We usually use 100 ng/mL M-CSF to mature them for 6 days. Thats the only way we were able to make them survive actually.

For GM-CSF you can use 5 units/mL. But it is possible one of your reagents or materials during purification is contaminated, for example LPS.

Just let them sit for 6 days with M/CSF or GM/CSF and get rid of the floaty ones after that. Adherence yield will really vary between donors. Recover the sticky ones by scraping and distribute equally in different wells at the required density. They will all readhere very quickly. Good luck.

Hello Stefan,

I am currently doing lot of these experiments. The procedure which i follow is: We isolate Monocytes using CD14 magnetic selection and plate them. They usually adhere in a couple of hours but sometimes during the differentiation they seem to be floating and this is common as they always attach n de-attach. Our medium contains 10% Human serum, 1% P/S, 0.05% Glutamine. The media change is on 4th and 7th day. We get the fully differentiated macrophages on Day 11. If you add MCSF or GMCSF the the Macrophages should be ready by Day7. On Day 4 we will centrifuge the medium in the wells, collect the cells and put them back into the new medium where as on Day 7 we will directly aspirate the medium and add new medium. In my experience, i never found "completely" adherent cells until Day7 (some are floating and some are adherent) and all the floating cells on Day 7 are considered as dead.

Good luck.

cheers,

Aravind

This is a common problem if you do not take steps BEFORE the first adherence to enrich the monocytes first, as they will never achieve the necessary cell density to form a lawn - there is a sort of threshold you need to cross where monocytes are packed in close to each other and then, BINGO: like magic, they form a macrophage lawn. Prior enrichment can be with a magnetic selection step or all sorts of ploys to deplete other cell types and platelets (which compete for adherence space). If you let me know what kind of plates or wells you are using, what cell numbers you need and what the readout entails then I can advise further.

Even tough you are using autologous serum, there may not be enough cytokines present to promote differentiation. Also, monocytes without enough cytokine present are not viable for very long. Note, if you use MCSF you are already polarizing the macrophages to a more M2 phenotype. Using GMCSF directs polarization more toward M1 phenotype. Some papers describe using small amounts of each, however, I have not been able to sucessfully do this while preventing directed polarization. The issue is what you plan to do with the macrophages.

Monocytes can differentiate and lawn into macrophages in the presence of autologous serum without exogenous growth factors, but it is contingent on achieving the necessary cell density in the first place, as explained above.

Definately with Aravind. Using a magnetic bead isolation procedure (e.g. Miltenyi Biotec) to either negatively or positively select the cells of interest will allow you to achieve consistency in your generation of MDMs. Use commercially available human serum where you can to decrease inter-individual variability. I think MCSF/GMCSF is optioonal really if you are using human serum but the MCSF will speed it up and give you some really nice cells. Look up Lester Kobzik in Experimental Lung research since they thought GMCSF gave a more alveolar macrophage phenotype- depending on your requirements...adam

Robert has given the secret basically. Thats the way Siamon Gordon's lab used to do it before when they didn't supplement with GM-CSF or M-CSF (or even use MACS beads). The one thing I'd add if you're not doing it is to definitely use non tissue culture treated plates. 12 wells are brilliant to get a good density going at 1-2 million monocytes/ml.

We usually use Percoll to separate the PBMC's from the granulocytes etc. and then seed at a density ~ 1 million/ml in 24-well Nunc plates. We usually only use magnetic isolation to isolate things such as eosinophils which are present in even lesser quantities but that is something we could definitely try.

Indeed, for previous studies my supervisor used to simply use autologous serum and everything was fine. However, now they just don't seem to like us and it isn't working.

Thank you for the heads up on the macrophage phenotypes. An alveolar macrophage-like phenotype would prove beneficial for my main study.

Thank you everybody for the useful information, especially the timings and stimulating concentrations.

I do agree with the magnetic enrichment step to increase the monocyte population before adherence step. But if you do not have the commercial you could try my protocol: I have achieved good and high quality monocyte derived macrophages population without using the magnetic enrichment. I isolate the PBMCs by gradient centrifugation and purify the population (remove platelets) with 5-7 washes with PBS. The PBMC pellet is then suspended in 1640 RPMI, transferred to T75 tissue culture flask and incubated for 2hr at 37oC with 5% CO2 to allow monocyte adherence. After 2hr, remove the non-adherent population and replace with ice cold PBC and incubate on ice for 30min to detach the monocytes. Using cell counter e.g haemocytometer determine the number of monocytes you want to plate. Suspend the cells in GMCSF containing 1640 RPMI medium and transfer cells to 24 well plates, incubate at 37oC with 5% CO2 for 24hr. Replace the GMCSF containing medium and replace with 1640 RPMI with 1% Pen/Strep and 10% human serum (obtained commercially). Cells are maintained in 1640 RPMI with Pen/Strep and 10% human serum for 7 days with medium changed every 3 days. To achieve a population of 10^5 macrophages after 7 days of culture I plate 5x10^5 monocytes per well (of the 24 well plate) at day zero.

Only about half of the cells become adherent. When you study surface phenotype or gene expression, there is no difference between floating and adherent cells. I think they cycle between the two states. If you want more controlled conditions, you can use Nutridoma (serum-free). See

Cho H, Shashkin P, Gleissner C, Dunson D, Jain N, Lee J, Miller Y, Ley K. Induction of dendritic cell-like phenotype in macrophages during foam cell formation. Physiological Genomics 2007;29:149-60.

we have prepared monocyte derived macrophages in this manner with success. We have used Iscoves medium however, in place of RPMI and 10% pooled human serum (pool of about 12 donors). Using autologous serum to culture should be OK although adds another variable between experiments. Be careful with the density of plating: aim to achieve 3 milliion monocytes per 10 cm dish or equivalent (ie about 30 million PBMC). Adhere for 2 hours at 37oC in serum free medium as serum does inhibit attachment. Wash away the lymphocytes with 6-8 washes in warm PBS then add culture medium and change every 3 days until the cells differentiate into macrophages. The use of human serum will give a mixed population of macrophages. If you want to polarize your macrophages use medium containing foetal calf serum supplemented with either M-CSF or GM-CSF.

Autologous serum is a kind of critical factor to determine cell survival. Even the apoptosis of cultured primary cells is observed with allogenic serum. Although minimum cell survival is available in FBS or Horse serum, any survival is not sufficient within autologous serum.

We first plate our PBMCs in serum free medium and allow them to rest for 30-45 minutes. Then we change the medium to medium containing serum (gently swirl the plate and remove the medium). You can continue to treat the cells with complete medium for 7 days and some will adhere (you will have to wash thoroughly to remove the contaminating cells). However, we find the best results are obtained when the cells are supplemented with M-CSF - in this case, medium containing M-CSF is added after one day in complete medium (again, wash gently here). Medium changed every 2 days to medium with M-CSF and thorough washes should give you a nice monolayer with a decent purity.

We are currently culturing HIV infected monocytes to differentiate them into macrophages. We isolate the PBMC with Histopaque and culture the cells in RPMI-1640 w/ glutamax, pen/strep, 10% FBS (HEAT-INACTIVATED). After 2 hrs or less the monocytes are already attached to the plastic. We wash the adherent monocytes at least 4 times with fresh warmed medium and change the medium (half-exchanged) every 3 days. Once they touch the plastic they become activated and produce M-CSF. If you want to accelerate the differentiation, you can add M-CSF (20ng/ml).

If the monocytes have been frozen and thawed correctly this should not be  a problem: We have successfully cultured  human monocytes to monocyte-derived macrophages after they were frozen  in 90% serum/10% tissue culture grade DMSO at a density of 10^7 per mL.

Hi,

I realize this thread is quite old already and might not warrant any replies! We are currently trying to differentiate monocytes to to macrophages using autologous serum from the same donor. The general protocol we follow is as such:

1) standard PBMC isolation using Ficoll

2) Plating isolated PBMC fraction in a non TC plate for 30 min-1 hour to allow monocyte adhesion

3) Washing off adherent cells with DPBS

4) adding RPMI+ pen strep + 10-40% autologous human serum and culturing withour disturbance for 6 days.

However, within the first few days itself the cells begin to look pretty terrible and almost none survive and/or differentiate.

I was wondering what I could start with in terms of troubleshooting prior to resorting to magnetic isolation of monocytes or using any sort of growth factor within the medium.

I'd be very thankful for any suggestions

You have to add a monocyte enrichment step between steps 1) and 2). If you omit this step, the monocytes will be sparsely adherent, and spread out too far away from each other to be able to form into a macrophage lawn.

You can use a percoll separation step or a magnetic bead selection step to enrich for monocytes. This will thin out platelets (which compete for adhesion space) and enrich the monocytes so that they achieve the magical plating density needed to form a contiguous macrophage lawn.

Dear Prof. Landis,

Thank you so much for your response:

Additionally, could I ask whether it is absolutely necessary to add additional growth factors? We'd prefer not to do this for the moment, unless it is absolutely necessary for cell viability.

Additionally, since we have some stockpiles of plasma, I was wondering whether it is feasible to use plasma in place of the serum for our trial experiments? Or must it necessarily be heat inactivated serum

Thank you again

No, it is not necessary to add additional growth factors if you are including at least 10% autologous human serum (plasma will not substitute for serum).

Would it be possible to provide specific reccommendations for optimum seeding densities? In both 24 well and 96 well plate formats?

I like using 24 well plates at a relatively high seeding density: 5 x 106 cells/well (cells collected after the monocyte enrichment step). 24 well plates are convenient for washing of lawns, to remove non-adherent cells, easier than in 96 well plates. The technique is fully described in this paper:

Landis, R.C., Yagnik, D., Emons, V., Philippidis, P., Mason, J.C., and Haskard, D.O. “Safe disposal of inflammatory urate crystals by differentiated macrophages” Arthritis Rheum. 46 (2002), 3026-33

Thank you for all your answers they have been really informative.

Recently I applied many of the suggestions described in this forum and still encounter one problem, the monocytes I isolete and culture do not adhere well to tissue culture treated plate. The conditions are described below:

1.- Isolate PBMC form human blood using Ficoll.

2.- Isolate monocytes using CD14 positive selection kit from stem cell.

3.- Plated 1, 2, 3, 4, 5, 6 or 7 million CD14+ cells, in 5 mL of DMEM 10% FBS, 5% A/B human serum, 1% HEPES, 1% pen/strep, and 1% glutamine on 10 cm plates with 100 ng/mL of MCSF-1.

4.- Waited 3 days after culture at 37C and 5%CO2, and saw that in many of the plates were a lot of floating cells.

How can I enhance adherence of my isolated cells ?

Thank you very much

Can you describe in detail:

- the CD14 selection step

- the types of plates used

Dear Dr. Landis,

Thank you for your answer.

After isolating PBMC using Ficoll centrifugation we resuspend the cells in 1X DPBS, 2%FBS, 1mM EDTA, at 100.000.000 cell/mL and use the EasySep human CD14 selection kit (catalog #: 18058) from Stem cell. We follow the manufactor instruction.

We add 100uL of CD14 beads per 1 mL of cell suspention and incubate for 15 min at room temp.

Then we add half of that volume (50uL) of magnetic beads per mL of cells and we incubate for 10 min at room temp.

The kit is based on CD14 positive selection using antibody couple beads, which then are conjugated with magnetic beads. After conjugation the cells are separated using a magnet (immunomagnetic separation using the big easy magnet from stem cells, catalog #: 18001).

We do three 5 minutes incubation of the cells in the magnet to separate the cells.

After isolation with the kit we resuspend the cells, count and plate at the density desired in DMEM 10% FBS, 5% A/B human serum, 1% HEPES, 1% pen/strep, and 1% glutamine supplemented with 100 ng/mL of MCSF-1.

We use Nunclon delta surface cell culture 10 cm plate catalog #: 172931.

Thank you very much,

Matias

Dear Matias, I recommend switching from positive to negative selection in the monocyte enrichment step. Miltenyi does a nice neagtive selection kit that generates untouched monocytes, free of any attached magnetic particles. Try that first. There are also some possible changes to your incubation medium one could consider if problems persist.

Monocyte Isolation Kit II:

http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-reagents/microbeads-and-isolation-kits/monocytes-and-macrophages/monocyte-isolation-kit-ii-human.aspx

Dear Dr. Landis,

Thank you so much!

I will try negative selection

Dear Dr Landis,

Thank you so much for your help previously. We have now tried the CD14 straight from whole blood monocyte isolation kit from Miltenyi and have successfully managed to differentiate these to macrophages using 10% human serum in RPMI pen/strep. 500,000 cells per well of a 24 well plate seem to work fine

I now have some additional questions I was wondering you could help me with:

1) Detaching the macrophages from the plate is proving quite tricky. Cell recovery even after a 20 min incubation with Accutase was quite poor. Do you have any suggestions? I would like to avoid using something as harsh as trypsin as far as possible.

2) Did you ever use these differentiated macrophages for downstream applications including qPCR? How is the RNA yield and integrity and is there anything I should keep in mind? What is the minimum cell number needed would you say

Thank you very much for all your great suggestions

Hi Nayana,

For detaching macrophages remove media, add warm PBS-1mM EDTA for a couple of seconds to wash, and discard. Then add again warm PBS-1mM EDTA and leave the plate for ~3-5 min in incubator. Finally, tapping the plate and pipetting up and down a few times should complete the trick. Also, before centrifugation of the obtained cell suspension in a tube, make sure you add one volume of media to lessen the stress on cells.

Hi Ricardo,

Thanks for the response! I will give it a try. In principle, is it ok to also use Trypsin if my downstream applications are qPCR and western blotting?

Also standard PBS or without Mg/Ca??

Thanks a lot

Dear Nyana, glad to hear that you have a macrophage lawn! Yeh :-). That is the hard part over. Detaching cells with complete viability and good for downstream use such as qPCR is no problem.

Rinse the plate four times with room temperature PBS and then add ICE-COLD PBS containing 2.5mM EDTA (EDTA is difficult to get into solution and you will need to make a 0.5M stock solution at pH 8.0 in the first place). Place the culture dish/flask into the fridge or preferably ON ICE for 30 minutes. If you look at the cells under the microscope they will have rounded up or even detached by 30 minutes. Scrape off the cells with a rubber policeman and then place into a solution of ice-cold HBSS WITHOUT Mg2+ and Ca2+ but with 2% FCS. Use polypropylene tubes for all subsequent steps. The detached cells should be 100% viable and usable for downstream applications like RNA extraction, qPCR, flow cytometry etc. You can expect to harvest about 20 microg RNA per well from a 24 well plate lawned at 5 x 106 cells, as you have done.

Hello Dr. Landis

I am learning quite a bit from this forum, and was wondering if you could help me out. I am trying to generate monocyte derived macrophages without the use of growth factors. However, I am using fetal bovine serum instead if human serum in my experiments. Will this still work?

Thanks

No, that will not work. If you do not have growth factors you must use 10% Human Serum. Either autologous serum from the same donor, or pooled AB serum which is available commercially.

Thank you Dr. Landis! I will give that a try.

Dear Ivy,

We have generated MDMs using both methods; human serum and growth factors.

Here is the optimized method we have published according to the human serum:

http://zjrms.com/en/articles/7362.html

While using human serum, please be aware of the concentration of serum in the beginning of attachment process which should be kept at the lowest possible concentration. I wish you may find it helpful.

Wish u all the best.

Hello, I would like to take advantage of the comments to ask for suggestions.

I am culturing 500.000 monocytes CD14+ (negative selection) in plates of 24 wells, with 1 mL of RPMI (enriched with 10% FBS, glutamine, antibiotics, hepes and antimycotic),  for 5 days, with 50 ng/mL GM-CSF or M-CSF.  I change the medium between the 2 and 3 day (add again the differentiating factors). On the 5 day, I change the medium again and, in addition to treating the cells again with M-CSF or GM-CSF, I insert 20ng/mL IL-4 or 100ng/mL LPS + 20ng/mL IFN-y and incubate for another 2 days. At the end, I remove the medium for dosage. My macrophages are not producing relevant TNF-a concentrations. Additionally, the IL-6 concentrations are between 1-10 pg/mL.  Do you think it's a normal response? Do you have any suggestions about it?

I will follow some suggestions I received, such as incubating the monocytes in a final volume of 500 uL, decreasing the differentiation time or not inserting the growth factors on day 5, but I would like to know your opinion, please.

Additionally, do you think it is necessary to completely change the medium between 2 and 3 day? Do the medium changes necessarily have to be done after washing the well with PBS or I can just remove all the medium and insert a fresh one?

I thank you very much in advance,

Hi Caique Figueiredo

I've had similar issues with my monocyte-derived macrophages. I am culturing 500.000 monocytes CD14+ (positive selection) in plates of 24 wells, with 0.5 mL of RPMI (enriched with 10% FBS, glutamine, antibiotics, hepes and antimycotic),  for 6 days, with 50 ng/mL M-CSF.  I change the medium at day 3 (with 50ng/mL M-CSF again). Then on Day 6, I polarize macrophages with 100ng/mL LPS + 20ng/mL IFN-y + 20ng/mL M-CSF for 24 hours. When performing ELISA, I detect surprisingly low TNF-alpha and IL-6 cytokine levels. I am not sure why that's the case. I will keep you updated if I make any progress regarding this issue.

I would recommend not to remove all your media on Day 3 as you may still have non-adherent monocytes (which have not differentiated into adherent macrophages yet) floating in your supernatant. You can remove half your media (250uL if you have 0.5mL cRPMI in each well) and add fresh media (250uL) by multiplying by 2 the concentration of your M-CSF.