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

Summary

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.

Abstract

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.

Introduction

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.

In many cases, murine models are used to investigate the pathophysiology of specific diseases. However, studying macrophage biology using mouse models is accompanied by several shortcomings: (1) The proportion of leukocyte subset numbers (i.e. monocytes and granulocytes) in peripheral blood of mice or humans differs significantly suggesting different roles of monocytes in murine and human pathophysiology. (2) There are substantial differences in gene expression between murine and human peripheral blood monocytes suggesting substantial differences in their function during health and disease ⁴. (3) A number of markers that are used to identify murine monocytes and macrophages (F4/80, LyC, etc.) does not exist in human myeloid cells, making the transfer of findings in mouse models to the human situation rather difficult.

Thus, in order to increase our understanding of macrophage differentiation and heterogeneity in human disease, we need to make use of models working with human macrophages. Therefore, we here describe a model of human primary macrophage generation that is easy to use and allows study of human monocyte-derived macrophages in vitro under various conditions resulting in different macrophage polarization types. In several studies, we have used the in vitro model of monocyte-derived primary human macrophages to analyze macrophage biology and its potential relevance to human atherosclerosis ^5-7.

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.

Protocol

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 anticoagulant.
3. Isolate PBMC (histopaque) as follows:
   1. Prepare two sterile 50 ml tubes (no poly-styrene).
   2. Dilute blood from step 2 with PBS 1:1.
   3. Add 25 ml histopaque + 25 ml whole blood/PBS per tube. Make sure that histopaque and blood do not mix.
   4. Centrifuge at 400 x g for 30 min at RT, no brake.
   5. Aspirate plasma and discard.
   6. Aspirate opaque interface and pipette into clean 50 ml tube.
   7. Add 20 ml of 0.02% EDTA in PBS.
   8. Centrifuge at 250 x g for 5 min at 4 °C.
   9. Discard supernatant.
   10. Add 10 ml of 0.02% EDTA in PBS.
4. Count cells as follows:
   1. Mix well by vortexing for 10 sec.
   2. Take 200 μl cell suspension + 300 μl 0.02% EDTA in PBS + 500 μl of Trypan blue (200-500 cells/10 squares, otherwise change dilution factor).
5. Perform negative isolation of monocytes as follows:
   1. Centrifuge cells obtained in step 3.10 for 10 min, 120 x g. Discard supernatant.
   2. Wash cell pellet 2x by adding 10 ml of PBS + 0.02% EDTA and centrifuge for 10 min, 120 x g.
   3. Add 9 ml sterile water for 3 sec, add 1 ml 10X PBS.
   4. Wash once more with PBS + 0.02% EDTA and centrifuge 10 min, 120 x g.
   5. Count during centrifugation.
   6. Dilute in EasySep buffer at 5 x 10⁷/ml.
   7. Add 50 μl/ml monocyte enrichment cocktail.
   8. Incubate for 10 min at 4 °C.
   9. Vortex beads for 30 sec.
   10. Add 50 μl/ml beads.
   11. Incubate for 5 min at 4 °C.
   12. Fill up with EasySep buffer to complete volume of 2.5 ml. The solution now appears brownish.
   13. Put into magnet.
   14. Wait 2.5 min at RT. During this time non-monocytic cells bound to the magnetic bead will move to the tube wall and stick there.
   15. Pour buffer with non-binding monocytes into fresh sterile tube. This solution looks whitish.
   16. Wash once with PBS + 0.02% EDTA and centrifuge for 10 min, 120 x g.
6. Plate cells in a plastic dish or multiwall plate at a density of 0.5 x 10⁶/cm². Add 1 ml of culture media/1 x 10⁶ cells.
7. Culture cells under conditions of interest for 6-14 days.
8. Change media after 3 days by replacing 50% of the media with fresh media (see Table 1 for details).
9. After six days harvest cells for mRNA isolation, flow cytometry, Western blotting, etc.

Results

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...

Discussion

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...

Materials

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