In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

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.