Differentiation and Polarization of Monocyte-Derived Cells into Macrophage-Like Cells

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Differentiation and polarization of monocyte-derived cells

1. Use plastic adherence for isolation of monocytes. Briefly, seed freshly isolated peripheral blood mononuclear cells (PBMCs) in a 6-well culture plate at an appropriate concentration, e.g., 10 x 10⁶ PBMCs/well in 2 mL serum-free RPMI medium (Supplement RPMI 1640 with 1 mM sodium pyruvate, 2 mM L-glutamine and 10 mM HEPES) and incubate at 37 °C and 5% CO₂.
2. After 2–3 h, remove the non-adherent cells with a pipette and wash the wells 3 times with 1 mL serum-free medium. The attached cells are monocytes and comprise around 10% of the total PBMCs added to the well, i.e., 10⁶ monocytes retrieved from 10 x 10⁶ PBMCs added per well.
3. For macrophage differentiation, prepare a working solution containing 50 ng/mL GM-CSF or M-CSF for M1 and M2 macrophage polarization respectively, added in 2 mL of RPMI complete medium per well. Culture the cells in a 5% CO₂ incubator at 37 °C for 3 days.
4. On day 3, remove 1 mL of the cell culture medium carefully from the top layer of each well and supplement the cell cultures with 1 mL of fresh RPMI complete medium containing the double concentration of M-CSF or GM-CSF to obtain 50 ng/mL final concentration in the wells. Add the growth factors in a pre-made working solution of 100 ng/mL/well.
5. On day 6, add different stimuli for the last 18–20 h of cell differentiation to obtain fully polarized and mature M1 (interferon-γ; IFN-γ, and lipopolysaccharide; LPS (E. coli O55:B5)) or M2 (interleukin 4; IL-4) macrophages. For M1 polarization, prepare IFN-γ and LPS in RPMI complete medium (Supplement RPMI 1640 with 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, and 10% heat-inactivated fetal bovine serum (FBS)) and add 50 µL per well to obtain a final concentration of 50 ng/mL IFN-γ and 10 ng/mL LPS in the cell cultures. For M2 polarization, prepare IL-4 in RPMI complete medium and add 50 µL per well to obtain a final concentration of 20 ng/mL in the cell cultures.
6. For differentiation of M0 polarized macrophages, stimulate the cells with M-CSF only, without any additional cytokines (providing an M2-like phenotype).
7. Check the morphology of monocyte-derived cell cultures regularly with light microscopy to ensure that smaller monocytes are differentiated into larger macrophage-like cells. Also, monitor potential morphological differences between the M1 and M2 polarization, i.e., elongated and stretched M1 cells compared to M2 cells with a more rounded shape.