Name: culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages
Uploaded: 2016-06-30T15:00:00
Description: Watch this Scientific Journal Video about Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages at JoVE.com

The Department of Transfusion Medicine, Clinical Center, National Institutes of Health, provided elutriated monocytes. This work was supported by the Intramural Research Program, National Heart, Lung, and Blood Institute, National Institutes of Health.

Leukapheresis was carried out under a human subject&#39;s research protocol approved by a National Institutes of Health institutional review board.
1. Isolation and Cryopreservation of Monocytes
<ol>
	<li>Obtain mononuclear cells by leukapheresis of human donors, and enrich monocytes by continuous counter-flow centrifugation elutriation of mononuclear cells as described in the references 7,8. Obtain approximately 100 x 106 elutriated cells (approximately 80 - 90% monocy...

<ol>
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Biol. 30, 2022-2031 (2010).</li><li>Zhao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+receptor-independent+low+density+lipoprotein+uptake+and+cholesterol+accumulation+by+macrophages+differentiated+from+human+monocytes+with+macrophage-colony-stimulating+factor+(M-CSF).">Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF).</a> J. Biol. Chem. 281, 15757-15762 (2006).</li><li>Freeman, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCG1-mediated+generation+of+extracellular+cholesterol+microdomains.">ABCG1-mediated generation of extracellular cholesterol microdomains.</a> J. Lipid Res. 55, 115-127 (2014).</li><li>Kruth, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-independent+fluid-phase+pinocytosis+mechanisms+for+induction+of+foam+cell+formation+with+native+low-density+lipoprotein+particles.">Receptor-independent fluid-phase pinocytosis mechanisms for induction of foam cell formation with native low-density lipoprotein particles.</a> Curr. Opin. Lipidol. 22, 386-393 (2011).</li><li>Jin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCA1+contributes+to+macrophage+deposition+of+extracellular+cholesterol.">ABCA1 contributes to macrophage deposition of extracellular cholesterol.</a> J. Lipid Res. 56, 1720-1726 (2015).</li><li>Ong, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+cholesterol-rich+microdomains+generated+by+human+macrophages+and+their+potential+function+in+reverse+cholesterol+transport.">Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport.</a> J. Lipid Res. 51, 2303-2313 (2010).</li><li>Hashimoto, S., Yamada, M., Motoyoshi, K., Akagawa, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+macrophage+colony-stimulating+factor-induced+growth+and+differentiation+of+human+monocytes+by+interleukin-10.">Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10.</a> Blood. 89, 315-321 (1997).</li><li>Hashimoto, S. I., Komuro, I., Yamada, M., Akagawa, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+inhibits+granulocyte-macrophage+colony-stimulating+factor-dependent+human+monocyte+survival+at+the+early+stage+of+the+culture+and+inhibits+the+generation+of+macrophages.">IL-10 inhibits granulocyte-macrophage colony-stimulating factor-dependent human monocyte survival at the early stage of the culture and inhibits the generation of macrophages.</a> J. Immunol. 167, 3619-3625 (2001).</li><li>Lund, P. K., Joo, G. B., Westvik, A. B., Ovstebo, R., Kierulf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+monocytes+from+whole+blood+by+density+gradient+centrifugation+and+counter-current+elutriation+followed+by+cryopreservation:+six+years'+experience.">Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years' experience.</a> Scand. J. Clin. Lab. Invest. 60, 357-365 (2000).</li><li>Weiner, R. S., Normann, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+integrity+of+cryopreserved+human+monocytes.">Functional integrity of cryopreserved human monocytes.</a> J. Natl. Cancer Inst. 66, 255-260 (1981).</li><li>Hansen, J. 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Generating defined macrophage types can clarify some of the conflicting results obtained by investigators when studying macrophage biology. The use of various culture conditions and differentiation factors to generate primary human macrophages can lead to very different macrophage types, a fact that may not be appreciated by the researcher. For example, macrophages sometimes are generated from human monocytes using no serum, human serum alone, human serum supplemented with M-CSF, FBS alone, or FBS containing M-CSF or GM-...

Study of cultured macrophages is a useful model to understand the function of these cells in inflammation such as occurs in atherosclerotic plaques. When the research focus is on human diseases involving macrophages, it is useful to study primary human macrophages rather than non-human macrophages to avoid species differences. Also, the effects of cell transformation can be avoided by using primary macrophages rather than macrophage cell lines. For this purpose, macrophages differentiated from monocytes isolated from human blood serve as a means of obtaining primary human macrophages. 
Tissue macrophages may be either resident within tissues or may be derived from monocytes that migrate into the tissue and differentiate into macrophages 1. Two types of human monocyte-derived macrophages have been defined that differ not only in morphology but also gene expression and cell function 2. These two types are obtained from monocytes that are differentiated into macrophages in the presence of M-CSF + fetal bovine serum (FBS) and monocytes that are differentiated into macrophages in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) + FBS 2-6. In our experience (unpublished observation), use of human serum rather than FBS generates the GM-CSF type regardless of whether M-CSF or GM-CSF is included in the differentiation medium. M-CSF type macrophages tend to be more elongated than GM-CSF type macrophages, which resemble fried eggs in their morphology. Investigators should recognize that human M-CSF and GM-CSF monocyte-derived macrophages are not the same as so called M1 and M2 mouse bone marrow-derived macrophages 6.
During research using human monocyte-derived M-CSF differentiated macrophages, we experienced difficulty related to the availability of monocytes to initiate experiments and variation in the obtained cell densities of macrophages during differentiation of monocytes into macrophages. To overcome these problems, we have developed the following protocol in which monocytes are frozen until required for use, and monocytes are differentiated in bulk into macrophages that can then be removed from culture flasks and plated at desired cell densities to obtain more uniform cultures from experiment to experiment.

<table border="0" cellpadding="0" cellspacing="0" width="1086">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Cellbind 12-well culture plate</td>
			<td style="width:319px;">Corning</td>
			<td style="width:173px;">3336</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">CELLSTAR, T-75 flask, tissue culture treated</td>
			<td style="width:319px;">Greiner Bio-One North America</td>
			<td style="width:173px;">658157</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">RPMI 1640 culture medium</td>
			<td style="width:319px;">Cellgro Mediatech</td>
			<td style="width:173px;">15-040-CM</td>
			<td style="width:167px;">warmed to 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">L-Glutamine</td>
			<td style="width:319px;">Cellgro Mediatech</td>
			<td style="width:173px;">25-005-CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Fetal bovine serum</td>
			<td style="width:319px;">Gibco</td>
			<td style="width:173px;">16000-036</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">M-CSF</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">300-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">GM-CSF</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">300-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">IL-10&#160;</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">200-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">DMSO</td>
			<td style="width:319px;">Sigma</td>
			<td style="width:173px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Cryovial&#160;</td>
			<td style="width:319px;">Thermo Scientific&#160;</td>
			<td style="width:173px;">375418</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:428px;">DPBS without Ca2+ and Mg2+&#160;</td>
			<td style="width:319px;">Corning cellgro</td>
			<td style="width:173px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">0.25% Trypsin-EDTA&#160;</td>
			<td style="width:319px;">Gibco</td>
			<td style="width:173px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">50 ml polypropylene conical tube</td>
			<td style="width:319px;">Falcon</td>
			<td style="width:173px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Trypan Blue</td>
			<td style="width:319px;">Lonza</td>
			<td style="width:173px;">17-942E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Neubauer-improved bright light hemocytometer&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:319px;">Paul Marienfeld GmbH &#38; Co. KG</td>
			<td style="width:173px;">610031</td>
			<td>http://www.marienfeld-superior.com/index.php/counting-chambers/articles/counting-chambers.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">CoolCell LX cell freezing container</td>
			<td style="width:319px;">BioCision</td>
			<td style="width:173px;">BCS-405</td>
			<td>other freezing containers also should&#160; be adequate for this step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Liquid Nitrogen Storage System, CryoPlus 1</td>
			<td style="width:319px;">Thermo Scientific&#160;</td>
			<td style="width:173px;">7400</td>
			<td>any liquid nitrogen tank should be adequate</td>
		</tr>
	</tbody>
</table>

culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

The viability of fresh or cryopreserved monocytes was greater than 95% as determined with Trypan Blue staining 9. Figure 1 and Figure 2 compare at lower and higher magnifications, respectively, the progress of fresh and cryopreserved (i.e., frozen) monocyte differentiation into macrophages. Note that the fresh compared with cryopreserved monocytes show a subpopulation of differentiating monocytes that do not spread but...

Watch this Scientific Journal Video about Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages at JoVE.com

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

The overall goal of this procedure is to improve the availability of monocytes and to minimize the variation in obtained cell densities during MCSF induced differentiation of human blood monocytes into macrophages. This method can help answer key questions concerning human macrophage biology. The main advantage of this tactic is that human monocyte derived macrophage cultures can be readily obtained with reduced dependency on donor availability.In addition desired macrophage cell density and the homogeneous cultures can be consistently achieved. To begin this procedure, obtain the mononuclear cells by leukapheresis from human donors and then enrich the monocytes by continuous counterflow centrifugal elutriation of mononuclear cells. After that, count the cells using a hemocytometer.Next, centrifuge the monocytes in a 15 milliliter polypropylene tube for five minutes at room temperature. Then, remove the supernatant and gently re-suspend the cells in FBS followed by Dimethyl Sulfoxide to achieve a final concentration of 90%FBS, 10%Dimethyl Sulfoxide, and 50 times 10 to the six cells per milliliter. Afterward add one milliliter of cell suspension to an individual cryofile.Place the cryofiles in a cell freezing container, and transfer it to the minus 80 degrees Celsius freezer for 24 hours. After 24 hours, transfer the container to a liquid nitrogen cyrofile tank for long term storage. In this step, thaw a cyrofile of cells by briefly keeping it in a 37 degrees Celsius water bath.When the cell suspension has thawed about 70%remove it from the water bath immediately and transfer it to room temperature. After the cell suspension has been completely thawed, transfer one milliliter of the cyrofile contents to 50 milliliters of RPMI 1640 medium with two millimolar L-glutamine, 50 nanograms per milliliter M-CSF, 25 nanograms per milliliter IL-10, and 10%FBS at 37 degrees Celsius. Then, transfer 25 milliliters of monocyte suspension into two 75 square-centimeter plastic cell culture flasks.Incubate the cultures at 37 degrees Celsius for 48 hours. Remember do not change the medium until 48 hours;otherwise, the cells won't be well attached and will be rinsed away. After 48 hours, rinse the cultures three times with 10 milliliters of pre-warmed RPMI 1640 medium.Gently remove the culture medium to avoid dislodging any loosely attached cells. Subsequently, add complete medium and culture the cells for one week. Change the medium every two days until the monocytes differentiate and proliferates efficiently to become confluent.Now, rinse the differentiated macrophages in the flask three times with 10 milliliters of 37 degrees Celsius DPBS without calcium and magnesium. Subsequently, add 10 milliliters of 37 degrees Celsius 0.25%Trypson EDTA solution. Next, place the flask in a cell culture incubator for 10 to 15 minutes.Following this, approximately 90%of the macrophages should have rounded and detached. Then, add 10 milliliters of RPMI 1640 medium containing 10%FBS to stop trypsinization. If necessary, use a cell lifter to completely remove the cells.Sometimes in this procedure many macrophages remain attached. These macrophages can be removed following trypsinization by passing a cell lifter across the flask surface. Afterward, transfer the macrophage cell suspension from the flask into a 15 milliliter polypropylene tube.Centrifuge it for five minutes at room temperature and discard the supernatant. Then gently simply re-suspend the macrophages in one milliliter of complete medium. Based on the cell count, add additional complete medium to achieve the desired seeding cell density.Then, plate the macrophages in 1.5 milliliters of medium per well of a 12 well plate. One times 10 to the fifth cells in 1.5 milliliters of medium per well, usually produces a near confluent culture. Following seeding, incubate the macrophages overnight at 37 degrees Celsius to allow cell attachment before using them in experiments.This figure shows the face contrast microscopic images of cryopreserved fresh monocytes during their MCSF induced differentiation into macrophages at low magnification. The images of monocytes were taken on day three and seven of culture in flask, and on day eight, one day after their harvesting and re-plating in the 12 well culture plate. Shown here is the comparison of fresh versus cryopreserved monocytes used to produced macrophages at high magnification.The phase loosened vacuoles are macropinosomes. After watching this video you should have a good understanding of how to cryopreserve and later differentiate human monocytes with MCSF into macrophages. Subsequently, the macrophages can be cultured at desired cell densities for experiments.

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