In Vitro Differentiation of Human PBMC Derived Monocytes into M1 or M2 Macrophages in a Serum-free and Xeno-free Cell Culture Media

Figure 1. Protocol overview using PromoCell M1/M2 Macrophage Generation Medium DXF.

Macrophage Differentiation Protocol

1. Isolate mononuclear cells (day 0). Isolate fresh PBMCs from buffy coats using your routine protocol.
2. Analyze mononuclear cells (day 0). Count and analyze the isolated PBMCs for monocyte content, (e.g. using the FSC/SSC plot of a flow cytometer). Subsequently resuspend the cells at 100X10⁶ PBMCs/ml in Monocyte Attachment Medium (C-28051).
3. Let the monocytes attach (day 0). Plate freshly isolated PBMCs in an appropriate amount of Monocyte Attachment Medium (C-28051), e.g. 15 ml Medium per T-75 flask. Use a seeding density of 1 million/cm² for Mononuclear Cells with a monocyte content of ≥25% and 1.5 million/cm² for a monocyte content of <25%. Incubate for 1-1.5 hours at 5% CO₂ and 37°C in the incubator without any further manipulation.
4. Prepare the complete Macrophage Generation Medium DXF (day 0). Prepare the Macrophage Generation Medium DXF (C-28055, C-29056) by adding the thawed Supplement Mix aseptically to the Basal Medium. Swirl gently to obtain a homogeneous mixture. Then, add Cytokine Mix M1 or M2, respectively.
5. Wash the adherent cell fraction (day 0). By vigorously swirling the tissue culture vessel loosen non-adherent cells and aspirate them. Wash the adherent cells, i.e. monocytes, three times with warm Monocyte Attachment Medium (C-28051) by swirling the vessel and aspirating the supernatant.
6. Start the macrophage differentiation (day 0). Add an appropriate amount of complete M1- or M2-Macrophage Generation Medium DXF (C-28055, C-29056) to the cells, e.g. 20 ml per T-75 flask and incubate for 6 days at 37°C and 5% CO₂ without medium change.
7. Continue the differentiation process (day 6). Add another 50% to 75% by volume of fresh complete M1- or M2-Macrophage Generation Medium DXF (C-28055, C-29056) to the cells. Incubate the immature macrophages for another 3 days at 37°C and 5% CO₂.
8. Optional step: macrophage activation (day 7). For specific macrophage activation the whole volume of the culture is supplemented with adequate stimuli of the customers´ choice. Do not perform a medium change, just add the activation factors. Examples of macrophage activation by defined stimuli: Classically activated M1 macrophages can be generated by addition of IFN-γ (50 ng/ml) and LPS (10 ng/ml) to M1 macrophages. M2a-activation of M2 macrophages is achieved by 20 ng/ml IL-4. Supplementation with immune complexes and IL-1β or LPS will elicit M2b-activation, whilst IL-10, TGFβ or glucocorticoids lead to M2c-activation of M2-macrophages. An alternative type of M1 activated macrophage can be obtained by the activation of M2 macrophages with IFN-γ and LPS [2].
9. Medium change (day 9). Aspirate the medium including suspension cells and collect it in a centrifugation tube. Immediately, pipet fresh complete PromoCell Macrophage Generation Medium DXF (C-28055, C-29056) supplemented with appropriate cytokines/activation factors to the cells. Centrifuge the cells in the tube for 15 min at 350 x g at room temperature. Discard the supernatant and carefully resuspend the cells in a small amount of fresh medium. Combine the resuspended cells in the tube with the adherent cells in the fresh medium contained in the tissue culture vessel. Incubate till the next day at 37°C and 5% CO₂.
10. The macrophages are ready (day 10). The macrophages may now be used directly in the plates where they reside, e.g. when performing phagocytosis assays. Maintenance of the culture for up to three weeks by performing weekly medium changes with fresh complete Macrophage Generation Medium DXF (C-28055, C-29056) is possible.

Results

Figure 2. Morphology and phagocytic activity of human PBMC derived M1 macrophages. A) Phase bright images of M1 macrophages 24 hours after plating in PromoCell’s Macrophage Generation Medium DXF. B) PromoCell cryopreserved M1 macrophages ingest large numbers of fluorescently labeled E.coli. bacteria.

Figure 3. Morphology and phagocytic activity of human PBMC derived M2 macrophages. A) Phase bright images of M2 macrophages 24 hours after plating in PromoCell’s Macrophage Generation Medium DXF. B) PromoCell cryopreserved M2 macrophages ingest large numbers of fluorescently labeled E.coli. bacteria.

Figure 4. Flow cytometry analysis of day 10 M1 and M2 macrophages generated in the PromoCell Macrophage Generation Medium DXF. Fresh peripheral blood mononuclear cells (PBMCs) were plated in the Monocyte Attachment Medium. The purified monocytes were differentiated for 10 days without performing the optional activation step. M1 macrophages exhibit the CD68+ (99% positive) and CD80+ (83% positive) marker profile while M2 macrophages exhibit a CD68+ (99% positive) and CD163+ (95% positive) marker profile.

References

1. Murray PJ, Wynn TA. 2011. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol. 11(11):723-737. https://doi.org/10.1038/nri3073

2. Murray PJ, Wynn TA. 2011. Obstacles and opportunities for understanding macrophage polarization. Journal of Leukocyte Biology. 89(4):557-563. https://doi.org/10.1189/jlb.0710409

3. Khramtsova G, Liao C, Khramtsov A, Li S, Gong C, Huo D, Nanda R. 2009. The M2/Alternatively Activated Macrophage Phenotype Correlates with Aggressive Histopathologic Features and Poor Clinical Outcome in Early Stage Breast Cancer.. https://doi.org/10.1158/0008-5472.sabcs-09-107

4. Menzies FM, Henriquez FL, Alexander J, Roberts CW. Sequential expression of macrophage anti-microbial/inflammatory and wound healing markers following innate, alternative and classical activation. 160(3):369-379. https://doi.org/10.1111/j.1365-2249.2009.04086.x