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Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Published: June 12th, 2013



1Department of Cardiology, University of Heidelberg

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

Monocyte-derived macrophages represent an important cellular component of the innate immune system and contribute to many acute or chronic inflammatory processes 1. Macrophages play an important role in many inflammatory diseases like atherosclerosis or cancer 2. Macrophages show a high degree of plasticity and are able to assume different phenotypes depending on the local micromilieu 3. Thus, studying macrophage differentiation and heterogeneity is essential for increasing our knowledge of the pathophysiology of many diseases and to allow identification of novel therapeutic targets and development of novel therapies.

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1. Protocol

  1. Prepare Buffers as follows:
    1. Prepare buffer for PBMC isolation: "Wash buffer" = 0.02% EDTA in PBS (use 0.5 M EDTA).
    2. Prepare buffer for monocyte isolation: "MACS rinsing buffer" = 0.5% BSA (250 mg) + 2 mM EDTA (200 μl) + PBS (50 ml). Degas the buffer.
    3. Prepare buffer for FACS staining and storage of cells: "FACS buffer" = 10% FCS in PBS and "fixation buffer" = 1% PFA in PBS.
  2. Draw 30 ml whole blood from a forearm vein. Use EDTA as anticoagul.......

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Using the protocol described above, we routinely obtain 25.1 x 106 ± 2.2 x 106 monocytes/100 ml blood (average ± standard error from 26 independent experiments, Figure 1A). Monocyte purity as determined by flow cytometric staining for CD14 is routinely greater than 95% (97.1 ± 0.4%, average ± standard error from 3 independent experiments, Figure 1B). Cell viability of freshly isolated monocytes as determined by trypan blue staining is rou.......

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Monocyte-derived macrophages represent the key cell type of the innate immune system. They play an important role in many inflammatory diseases including atherosclerosis or cancer 2. Thus, studying macrophage biology is essential for increasing our knowledge on the pathophysiology of many diseases and to allow development of novel therapies.

Many studies apply of mouse models overexpressing or lacking certain genes of interest. In the case of monocyte-derived macrophages, this seems.......

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We thank Nadine Wambsganss for excellent technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (GL599/1-1) and in part by a grant from the Innovation Fund FRONTIER (University of Heidelberg) to C.A.G.


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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalog Number Comments
50 ml centrifuge tube (sterile) Fisher 055398  
D-PBS (1X), liquid (no calcium or magnesium) Invitrogen 14190-250  
EDTA Sigma T9285  
BSA Sigma A-8806  
FCS Invitrogen    
EasySep Human Monocyte Enrichment Kit StemCell Technologies 19059  
EasySep Magnet StemCell Technologies 18000  
FACS tubes Fisher 352008  
Macrophage-SFM (1X) Invitrogen 12065-074  
Penicillin-streptomycin Sigma P-4458  
Nutridoma-SP Roche 11011375001  
human M-CSF 10 μg Peprotech 300-25  
Cell Culture Plates 6-well Fisher 07-200-80  

  1. Gordon, S., Taylor, P. R. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953-964 (2005).
  2. Gordon, S., Mantovani, A. Diversity and plasticity of mononuclear phagocytes. European Journal of Immunology. 41, 2470-2472 (2011).
  3. Martinez, F. O., Sica, A., Mantovani, A., Locati, M. Macrophage activation and polarization. Front. Biosci. 13, 453-461 (2008).
  4. Ingersoll, M. A., et al. Comparison of gene expression profiles between human and mouse monocyte subsets. Blood. 115, 10-19 (2010).
  5. Gleissner, C. A., Sanders, J. M., Nadler, J., Ley, K. Upregulation of aldose reductase during foam cell formation as possible link among diabetes, hyperlipidemia, and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 28, 1137-1143 (2008).
  6. Gleissner, C. A., et al. CXCL4 downregulates the atheroprotective hemoglobin receptor CD163 in human macrophages. Circ. Res. , (2009).
  7. Gleissner, C. A., Shaked, I., Little, K. M., Ley, K. CXC chemokine ligand 4 induces a unique transcriptome in monocyte-derived macrophages. J. Immunol. 184, 4810-4818 (2010).
  8. Martinez, F. O., Gordon, S., Locati, M., Mantovani, A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: New molecules and patterns of gene expression. J. Immunol. 177, 7303-7311 (2006).
  9. Verreck, F. A. W., et al. Human il-23-producing type 1 macrophages promote but il-10-producing type 2 macrophages subvert immunity to (myco)bacteria. Proceedings of the National Academy of Sciences of the United States of America. 101, 4560-4565 (2004).
  10. Rey-Giraud, F., Hafner, M., Ries, C. H. In vitro generation of monocyte-derived macrophages under serum-free conditions improves their tumor promoting functions. PLoS One. 7, e42656 (2012).
  11. Boyle, J. J., et al. Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype. American Journal of Pathology. 174, 1097-1108 (2009).
  12. Cho, H. J., et al. Induction of dendritic cell-like phenotype in macrophages during foam cell formation. Physiol Genomics. 29, 149-160 (2007).
  13. Vogt, G., Nathan, C. In vitro differentiation of human macrophages with enhanced antimycobacterial activity. The Journal of Clinical Investigation. 121, 3889-3901 (2011).
  14. Mikita, T., Porter, G., Lawn, R. M., Shiffman, D. Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus. Journal of Biological Chemistry. 276, 45729-45739 (2001).
  15. Qin, Z. Y. The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature. Atherosclerosis. 221, 2-11 (2012).
  16. Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K. B., Dockrell, D. H. The identification of markers of macrophage differentiation in pma-stimulated THP-1 cells and monocyte-derived macrophages. PLoS One. 5, e8668 (2010).
  17. Wahl, L. M., Wahl, S. M., Smythies, L. E., Smith, P. D. Isolation of human monocyte populations. Current Protocols in Immunology. , (2001).
  18. Waldo, S. W., et al. Heterogeneity of human macrophages in culture and in atherosclerotic plaques. Am. J. Pathol. 172, 1112-1126 (2008).
  19. Martín-Fuentes, P., et al. Individual variation of scavenger receptor expression in human macrophages with oxidized low-density lipoprotein is associated with a differential inflammatory response. The Journal of Immunology. 179, 3242-3248 (2007).

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