I have prepared human monocyte derived macrophages using 10%FBS, with either GMCSF or MCSF added, for several years now. I am currently trying the protocol using HuAB serum and no added cytokines. Has anyone had experience making them this way? I am getting some odd phenotype results with surface marker staining.
We now use human AB serum to differentiate our MDMs. Before that, we used 5% autologous inactivated plasma and we also used 10% FBS. We have much less variability with human AB serum and we recover a good amount of live MDMs. We add recombinant human M-CSF at 25ng/ml but we also did some studies of macrophage polarization in which we did not use M-CSF at all. In our hands, they behave as MDMs should. What kind of surface marker problem do you have?
I have also found great variability in polarization between donors making statistics extremely difficult.
My suggestion is that the primary cell culture with species specific serum is the main obtacle in the cell therapy preparation. Any success in the culture for more than one months (Over 4 week) will be a great contribution for human life..
Do someone know how much the monocyte/macrophage multiplies with this treatment??
They do not multipy. They are just differentiated to macrophages and then can be polarized.
Based on my expectation, the cells of monocyte would not proliferate.
@Benjamin and Juliette,
While there is general consensus that monocytes have limited proliferation capacity, some subpopulations of blood monocytes can proliferate and even "terminally differentiated" macrophages have been shown to be able to proliferate in vivo under certain conditions (TH2 inflammation).
http://www.biomedcentral.com/content/pdf/ar2616.pdf
http://www.jleukbio.org/content/66/6/953.abstract
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128495/
For the blood monocytes, you are right. They have discovered that RANK-RANKL signals can induce monocyte proliferation while pushing them towards osteoclasts. I wonder what OTHER signals could push blood monocytes to proliferate...
For the macrophages, it is related to the type of activation signal in vivo.
@saroush Sharbati do you have links to papers/articles that discuss the serum free method of preparation? We do macrophage/monocyte polarization in my laboratory and see extremely variable results, similar to what Doris mentioned. If this removed some of that variability that would be a huge step forward for my lab.
Before the beginning of cell culutre in serum free media, the purpose of cell culture should be reviewed. I wonder whether the cells growing in serum free media is similar to the cells in animal (or in vivo) is the primary purpose. The cells in serum free media may be very different cell feature or functions from the cells in media carrying serum
You may also be getting dendritic cells which will give all sorts of interesting results.
And I would like to suggest the problem in the cell culture with allogenic serum. Lots investigation showed that the cells feeded the media carrying allogenic serum can not be maintained more than 4 weeks
I have tried using HuAB without adding cytokines and have not been successfull in maintaining cells long term. Supposedly, HuAB should have the needed cytokines, however, even when adding low levels of MCSF+GMCSF, the cell viability is greatly reduced., the FSC vs. SSC plots of these cells do not look like those with FBS, and staining patterns are quite variable. I have tried reproducing procedures from the literature with and without added cytokines and have beeen unable to get the reported results. When adding cytokine to FBS cells, you are driving them to M1 or M2 macrophages. Theoretically growing in HuAB should produce what is called an M0 which would have a different phenotype. I think this is an issue not easily resolved and many of us have been frustrated with. Also note that many of the papers from the Italian group actually grew tihe cells under non-adherent conditions which is a whole other issue.
Well, although that is not the issue to be easily resolved, that is important issue. The cells to be used for cellular therapy will meet allogenic or autonomic serum. When the cells are prepared not to survive within allogenic or autogenic serum, the cells used for cell therapy will be highly useful. The injected cells will die soon.
similar question, possible to culture monocytes with human plasma instaed of 5%FCS to harvest macrophages?
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