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.

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

<ol>
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The authors do not have to disclose any conflict of interest.

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...

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.

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

an in vitro model to study heterogeneity of human macrophage differentiation and polarization

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.

Using the protocol described above, we routinely obtain 25.1 x 106 &plusmn; 2.2 x 106 monocytes/100 ml blood (average &plusmn; 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 &plusmn; 0.4%, average &plusmn; standard error from 3 independent experiments, Figure 1B). Cell viability of freshly isolated monocytes as determined by trypan blue staining is rou...

Watch this Scientific Journal Video about An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization at JoVE.com

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

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.

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

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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Immunology and Infection

18.5K Görüntüleme.  University of Heidelberg. Monosit türevli histiositlerinden doğuştan gelen bağışıklık sisteminin önemli bir hücrelerdir. Burada, kullanımı kolay tarif In vitro modeli. Gradyan santrifüj, negatif boncuk izolasyon ve belirli bir hücre kültürü koşulları kullanılarak, monosit türevli histiositlerinden fenotipik ve fonksiyonel çalışmalar için oluşturulabilir.