This article details the process of culturing human monocytes and inducing their differentiation into macrophages in vitro. It highlights the roles of GM-CSF and M-CSF in promoting M1 and M2 macrophage phenotypes, respectively.
Monocytes are immune cells that differentiate into macrophages and antigen-presenting dendritic cells to protect against infection.
To culture monocytes and induce their differentiation in vitro, begin by taking a suspension of primary human monocytes in a suitable culture medium. Add the cell suspension into the wells of a culture plate. Incubate at physiological conditions to allow the cells to adhere.
Add differentiation-inducing cytokines — granulocyte-macrophage colony-stimulating factor, or GM-CSF, to one well and macrophage colony-stimulating factor, or M-CSF, to another. Incubate for an appropriate duration.
GM-CSF binds to its heterodimeric receptor on the cells and induces the differentiation of the monocytes. GM-CSF binding results in the activation of intracellular signaling pathways, leading to the expression of genes for priming into an M1 macrophage-like phenotype.
The M1-like cells — rounded cells with a large cytoplasmic volume — are pro-inflammatory and assist in the defense against infection.
In the other well, the M-CSF binds to its homodimeric receptor on the monocytes and induces the differentiation of the cells. M-CSF binding results in the activation of intracellular signaling pathways different from GM-CSF, leading to the expression of genes for priming into an M2 macrophage-like phenotype.
The M2-like cells — with an elongated shape — are anti-inflammatory and exhibit tissue remodeling and repair functions.
The differentiated macrophage cells are ready for further downstream assays.
After counting, re-suspend the isolated monocytes at a 1 times 10 to the sixth cells per milliliter of 37 degrees Celsius serum-free RPMI 1640 medium supplemented with antibiotics, and plate 1 milliliter of re-suspend cells into 35-millimeter plates in a biosafety cabinet. Then, place the plates in the cell culture incubator for 30 to 60 minutes to allow the cells to adhere to the well bottoms.
To prime for macrophages with an M1-like phenotype, add 100 microliters of fetal bovine serum containing 25 nanograms per milliliter of GM-CSF to each plate. To prime for macrophages with an M2-like phenotype, add 100 microliters of fetal bovine containing 50 nanograms per milliliter of M-CSF to each plate.
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