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Immunology and Infection

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

Published: June 12th, 2013

DOI:

10.3791/50332

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  

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