Name: culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages
Uploaded: 2016-06-30T15:00:00.000+00:00
Duration: 6 min 46 s
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% monocytes) in elutriation buffer specified by the manufacturer. Count cells using a hemocytometer.</li>
	<li>Centrifuge monocytes in a 15 ml polypropylene tube at 300 x g for 5 min at room temperature.</li>
	<li>Remove the supernatant and gently resuspend the cells in FBS followed by dimethyl sulfoxide to achieve a final concentration of 90% FBS/10% dimethyl sulfoxide and 50 x 106 cells/ml.</li>
	<li>Add each ml of cell suspension to an individual cryovial.</li>
	<li>Place the cryovials in a cell freezing container and transfer to a -80 &#176;C freezer for 24 hr before transferring to a liquid nitrogen cryovial storage tank for long-term storage.</li>
</ol>
2. Differentiation of Monocytes into Macrophages
<ol>
	<li>Thaw a cryovial of cells by quickly transferring to a 37 &#176;C water bath and then immediately removing when the cell suspension has thawed about 70%.</li>
	<li>Immediately upon complete thawing at room temperature, transfer the 1 ml cryovial contents into 50 ml of 37 &#176;C warmed Roswell Park Memorial Institute (RPMI) 1640 medium with 2 mM L-glutamine, 50 ng/ml M-CSF, 25 ng/ml interleukin-10 (IL-10), and 10% FBS (complete medium).</li>
	<li>Transfer 25 ml of monocyte suspension into each of two 75 cm2 plastic cell culture flasks.</li>
	<li>Incubate cultures in a 37 &#176;C cell culture incubator with 5% CO2 / 95% air for 48 hr.</li>
	<li>Rinse the cultures 3 times with 10 ml RPMI 1640 medium (pre-warmed to 37 &#176;C), gently removing the culture medium to avoid dislodging any loosely attached cells.</li>
	<li>Following rinsing, add fresh complete medium, changing medium every 2 days until monocytes differentiate and proliferate sufficiently to become confluent. This requires about one week of culture.</li>
</ol>
3. Harvesting Macrophages to Initiate Experiments
<ol>
	<li>Rinse differentiated macrophages in flask 3 times with 10 ml pre-warmed 37 &#176;C Dulbecco&#39;s phosphate-buffered saline without Ca2+ and Mg2+ before adding 10 ml&#160;pre-warmed 37 &#176;C 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution.</li>
	<li>Place flask in cell culture incubator for 10 - 15 min at which time approximately 90% of the macrophages should have rounded and detached. Verify this by microscopic examination of the flask.</li>
	<li>Add 10 ml of RMPI 1640 medium containing 10% FBS to stop trypsinization.</li>
	<li>Transfer the macrophage cell suspension from the flask into a 50 ml polypropylene tube, centrifuge 300 x g for 5 min, and discard the supernatant.</li>
	<li>Gently resuspend the macrophages into 1 ml of complete medium.</li>
	<li>Mix 10 &#181;l of macrophage cell suspension with 10 &#181;l Trypan Blue solution and determine macrophage cell number and viability (typically &#62; 95%) using a hemocytometer.</li>
	<li>Based on the cell count (usually about 15 - 18 x 105 macrophages), add additional complete medium to achieve the desired seeding cell density. Note: 1 x 105 macrophages in 1.5 ml medium per well of a 12-well plate will produce a near-confluent culture.</li>
	<li>Following seeding, incubate macrophages overnight in a 37 &#176;C cell culture incubator with 5% CO2 / 95% air to allow cell attachment. Then, begin desired experiments.</li>
</ol>

<ol>
	<li>Dey, A., Allen, J., Hankey-Giblin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+polarization+of+macrophages+in+inflammation:+blood+monocytes+versus+tissue+macrophages.">Ontogeny and polarization of macrophages in inflammation: blood monocytes versus tissue macrophages.</a> Front. Immunol. 5, 683 (2014).</li><li>Waldo, S. W., 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=Heterogeneity+of+human+macrophages+in+culture+and+in+atherosclerotic+plaques.">Heterogeneity of human macrophages in culture and in atherosclerotic plaques.</a> Am. J. Pathol. 172, 1112-1126 (2008).</li><li>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=Functional+heterogeneity+of+colony-stimulating+factor-induced+human+monocyte-derived+macrophages.">Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.</a> Int. J. Hematol. 76, 27-34 (2002).</li><li>Akagawa, K. 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=Functional+heterogeneity+of+colony-stimulating+factor-induced+human+monocyte-derived+macrophages.">Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.</a> Respirology. 11 Suppl, S32-S36 (2006).</li><li>Fleetwood, A. J., Lawrence, T., Hamilton, J. A., Cook, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(CSF)+and+macrophage+CSF-dependent+macrophage+phenotypes+display+differences+in+cytokine+profiles+and+transcription+factor+activities:+implications+for+CSF+blockade+in+inflammation.">Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation.</a> J. Immunol. 178, 5245-5252 (2007).</li><li>Lacey, D. C., 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=Defining+GM-CSF-+and+macrophage-CSF-dependent+macrophage+responses+by+in+vitro+models.">Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models.</a> J. Immunol. 188, 5752-5765 (2012).</li><li>Strasser, E. F., Eckstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+leukocyte+collection+and+monocyte+isolation+for+dendritic+cell+culture.">Optimization of leukocyte collection and monocyte isolation for dendritic cell culture.</a> Transfus. Med. Rev. 24, 130-139 (2010).</li><li>Kim, 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=Monocyte+enrichment+from+leukapheresis+products+by+using+the+Elutra+cell+separator.">Monocyte enrichment from leukapheresis products by using the Elutra cell separator.</a> Transfusion (Paris). 47, 2290-2296 (2007).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr. Protoc. Immunol. Appendix 3 (Appendix 3B), (2001).</li><li>Anzinger, J. J., 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=Native+low-density+lipoprotein+uptake+by+macrophage+colony-stimulating+factor-differentiated+human+macrophages+is+mediated+by+macropinocytosis+and+micropinocytosis.">Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.</a> Arterioscler. Thromb. Vasc. 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. 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=Retention+of+phagocytic+functions+in+cryopreserved+human+monocytes.">Retention of phagocytic functions in cryopreserved human monocytes.</a> J. Leukoc. Biol. 57, 235-241 (1995).</li><li>Seager Danciger, J., 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=Method+for+large+scale+isolation,+culture+and+cryopreservation+of+human+monocytes+suitable+for+chemotaxis,+cellular+adhesion+assays,+macrophage+and+dendritic+cell+differentiation.">Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation.</a> J. Immunol. Methods. 288, 123-134 (2004).</li><li>Jin, X., Xu, Q., Champion, K., 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=Endotoxin+contamination+of+apolipoprotein+A-I:+effect+on+macrophage+proliferation--a+cautionary+tale.">Endotoxin contamination of apolipoprotein A-I: effect on macrophage proliferation--a cautionary tale.</a> Atherosclerosis. 240, 121-124 (2015).</li></ol>

The authors have nothing to disclose.

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-CSF. As reported previously and as we have also observed, monocytes cultured for a week without serum or with FBS without added differentiation factors do not remain viable 3. As mentioned above, monocytes cultured with human serum alone or supplemented with M-CSF generate macrophages that show the &#34;fried egg&#34; morphology characteristic of monocytes differentiated with FBS plus GM-CSF. Thus, in the presence of human serum the M-CSF type macrophage showing a more elongated morphology is not obtained even though monocytes have been differentiated with M-CSF.
Interestingly, we have observed that differentiation of monocytes with FBS containing both M-CSF and GM-CSF, generates both the M-CSF elongated and GM-CSF &#34;fried egg&#34; morphological phenotypes in the same culture. This suggests that distinct monocyte subpopulations give rise to these two macrophage types. Previous studies have shown that M-CSF and GM-CSF differentiation factors affect not only the morphology of the macrophage, but also influence gene expression and cell function, which is substantially different for these two macrophage types 2-6. Immunostaining for the macrophage marker CD68 in conjunction with CD14 that is expressed by M-CSF type macrophages and 25F9 that is expressed by GM-CSF macrophages can be used to confirm the macrophage type as shown previously 2. These two macrophage types may be especially relevant to the study of atherosclerosis where different macrophage types occur in atherosclerotic plaques and can show differences in cholesterol metabolism and expression of inflammation mediators 2,12,13. For example, M-CSF type macrophages are unique in that these macrophages deposit cholesterol into the extracellular matrix when the macrophages are enriched with cholesterol 12,14,15.
In the protocol presented, during culture with M-CSF, IL-10 has been routinely added as described previously 16. This cytokine promotes the growth and differentiation of the macrophages and produces a more homogeneous elongated morphology of the macrophages. In addition, IL-10 inhibits GM-CSF-dependent monocyte survival by inhibiting the signaling events induced by GM-CSF 17. However, investigators should consider that IL-10 may affect other cell responses under study, and therefore, may wish to omit IL-10. Before using the current protocol employing proliferation and differentiation of the monocytes in a flask, macrophage cultures were initiated by directly plating elutriated monocytes into culture wells and differentiating them for 7 days prior to initiating experiments. However, even with the addition of IL-10, some of these macrophage cultures showed small numbers (usually less than 10%) of &#34;fried egg&#34; GM-CSF type macrophages contaminating the elongated M-CSF type macrophages. With the current protocol, GM-CSF type macrophage contamination does not occur for reasons that are not clear. However, it is not because GM-CSF macrophage type precursor monocytes are lost during freezing, as culturing the cryopreserved monocytes with FBS + GM-CSF produces excellent GM-CSF type cultures (see Figure 4).
The described protocol utilizes cryopreservation of monocytes before their differentiation into macrophages. Monocyte cryopreservation has been previously shown not to affect monocyte function and is commonly employed 18-21. In this regard, we have found that macrophages generated by the protocol above deposit cholesterol into the extracellular space similar to what we have described previously with non-cryopreserved monocytes that were cultured directly into macrophages 12. With monocyte cryopreservation, monocytes are always available to initiate experiments, independent of when the monocytes can be obtained from a donor. While it is not essential to cryopreserve the monocytes before proceeding with the protocol, cryopreservation did produce more homogeneous macrophage cultures in that all the macrophages were more spread rather than being both rounded and spread as was the case when fresh monocytes were used to initiate cultures. We have successfully generated macrophage cultures from monocytes that were kept frozen up to 6 months, (the longest time tested). On the other hand, seeding of cryopreserved macrophages into culture wells for experiments did not produce adequate macrophage cultures.
Critical steps in the protocol include immediately placing monocytes into the freezer following addition of dimethyl sulfoxide, and immediately transferring thawed monocytes to culture medium. Although it is common to remove DMSO by centrifuging frozen cells before they are seeded into culture, we did not find this was a necessary step in successful culture of the monocytes. However, investigators may need to do this if the concentration of residual DMSO (2 &#181;l/ml) is higher than what was used in the above protocol. It is important not to rinse monocytes until 48 hours following their seeding in culture flasks. Otherwise, some monocytes may be lost depending on their degree of adhesion to the culture surface. On the other hand, once the monocytes differentiate into macrophages, increased adhesion of the macrophages may require longer trypsin treatment to remove them, or a cell lifter can be employed to facilitate release after the initial trypsin treatment. If monocytes do not proliferate and differentiate, this could be due to some unusual characteristic of the monocyte donor, loss of activity of M-CSF, or due to the lot of FBS used. Also, of possible concern when cultures do not develop is contamination of reagents with endotoxin, an agent that inhibits macrophage proliferation 22.
Differentiating the cryopreserved monocytes in flasks with M-CSF allows their bulk 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.

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><tbody><tr><td>Cellbind 12-well culture plate</td><td>Corning</td><td>3336</td><td></td></tr><tr><td>CELLSTAR, T-75 flask, tissue culture treated</td><td>Greiner Bio-One North America</td><td>658157</td><td></td></tr><tr><td>RPMI 1640 culture medium</td><td>Cellgro Mediatech</td><td>15-040-CM</td><td>warmed to 37 &amp;deg;C</td></tr><tr><td>L-Glutamine</td><td>Cellgro Mediatech</td><td>25-005-CI</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16000-036</td><td></td></tr><tr><td>M-CSF</td><td>PeproTech</td><td>300-25</td><td></td></tr><tr><td>GM-CSF</td><td>PeproTech</td><td>300-03</td><td></td></tr><tr><td>IL-10&amp;nbsp;</td><td>PeproTech</td><td>200-10</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Cryovial&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>375418</td><td></td></tr><tr><td>DPBS without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Corning cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>0.25% Trypsin-EDTA&amp;nbsp;</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>50 ml polypropylene conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Trypan Blue</td><td>Lonza</td><td>17-942E</td><td></td></tr><tr><td>Neubauer-improved bright light hemocytometer</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG</td><td>610031</td><td>http://www.marienfeld-superior.com/index.php/counting-chambers/articles/counting-chambers.html</td></tr><tr><td>CoolCell LX cell freezing container</td><td>BioCision</td><td>BCS-405</td><td>other freezing containers also should&amp;nbsp; be adequate for this step</td></tr><tr><td>Liquid Nitrogen Storage System, CryoPlus 1</td><td>Thermo Scientific&amp;nbsp;</td><td>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 remain rounded. Phase-lucent vacuoles occur in the monocyte-derived macrophages in both conditions. These vacuoles represent macropinosomes characteristic of M-CSF type macrophages as previously described 10,11.
Cryopreserving macrophages after their harvesting from the flasks in which they were differentiated from monocytes did not produce successful macrophage cultures (Figure 3). Such cultures showed very sparse cell attachment possibly due to poor viability after cryopreservation.
The above protocol for producing M-CSF type macrophages does so without the presence of contaminating GM-CSF type macrophages that sometimes occurs when cultures are established directly from monocytes without their cryopreservation and differentiation to M-CSF type macrophages in flasks. However, cryopreserved monocytes differentiated with FBS + GM-CSF produce excellent GM-CSF type cultures (Figure 4).
<img alt="Monocyte cultures comparison; microscopy images; frozen vs. fresh over days; cell morphology results." src="/files/ftp_upload/54244/54244fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Comparison of fresh versus cryopreserved monocytes used to produce macrophages (low magnification). Phase-contrast microscopic images of cryopreserved (frozen) and fresh monocytes during their M-CSF induced differentiation into macrophages. Shown are monocytes during culture in flasks at 3 and 7 days, and on day 8, one day after their harvesting and replating into 12-well culture plates. Bar = 100 µm and applies to all. <a href="https://www.jove.com/files/ftp_upload/54244/54244fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Frozen vs. fresh monocytes, cell morphology comparison; microscope images, days 3, 7 in flask, day 8 plate." src="/files/ftp_upload/54244/54244fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Comparison of fresh versus cryopreserved monocytes used to produce macrophages (high magnification). Monocytes were cultured as described in Figure 1 legend and shown here at higher magnification. Phase-lucent vacuoles are macropinosomes (white arrows). Bar = 50 µm and applies to all. <a href="https://www.jove.com/files/ftp_upload/54244/54244fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell culture microscope image displaying diverse cell morphology, suitable for cell viability analysis." src="/files/ftp_upload/54244/54244fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Cryopreserved macrophages fail to produce adequate cultures. Fresh monocytes were seeded into a flask and differentiated 7 days with M-CSF as shown in Figures 1 and 2 above. Then, the differentiated macrophages were harvested from flasks and cryopreserved. Next, the cryopreserved macrophages were seeded into 12-well plates as described in the protocol for non-cryopreserved macrophages and cultured 1 day (day 8). Phase-contrast microscopic image of the 1-day culture shows that very few of the macrophages attach following their cryopreservation (compare to day 8 results for non-cryopreserved macrophages in Figure 1 above). Bar = 100 µm. <a href="https://www.jove.com/files/ftp_upload/54244/54244fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscope image of yeast cells; cell morphology study; high-contrast visualization for research." src="/files/ftp_upload/54244/54244fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Cryopreserved monocytes were also suitable to produce GM-CSF type monocyte-derived macrophages. Cryopreserved monocytes (25 x 106) were seeded into a flask and differentiated 8 days with 10% FBS containing 50 ng/ml GM-CSF. Phase-contrast image shows the typical "fried egg" morphology of GM-CSF type macrophages. Bar = 100 µm. <a href="https://www.jove.com/files/ftp_upload/54244/54244fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

29.1K Views.  National Institutes of Health. 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.

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

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.

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.

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

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient&#8217;s blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient&#8217;s blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these &#8220;educated&#8221; immune cells to the patient&#8217;s blood. Exosomes are nano-sized extracellular vesicles between 30&#8210;150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes

Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates ...

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s initiation and resolution phases, significantly affecting protective immunity and immune-mediated pathological injury. While tissue-resident macrophages are key players in maintaining homeostasis in a steady state, large amounts of monocytes are recruited from the peripheral blood into the tissue following damage or inflammatory insults. Monocyte-derived macrophages (M-DM) can differentiate into many dynamic subtypes, and their phenotypes and functions depend on the local tissue environment. 
To compare different stimuli or environmental conditions during M-DM differentiation and polarization, we standardized an in-vitro model of human nonpolarized M-DM M0 and some cardinal cytokine-polarized macrophages, IFN&#947;/LPS-derived M1, IL-4-derived M2a, and IL-10 or dexamethasone-derived M2c, to analyze skewing reprogramming of M-DM by flow cytometry and real-time PCR. We found that CD64, CD206, CD163, CD14, and MERTK can clearly discriminate unpolarized M0 and polarized M1, M2a, and M2c by flow cytometry. Moreover, we defined IRF1 and CXCL10 as specific genes for classical IFN&#947;/LPS-derived M1-, IRF4-, CCL22-, and TGM2-specific transcripts for IL-4-derived M2a, and the MERTK gene for dexamethasone-derived M2c. To summarize, our standardized M-DM protocol could give the cardinal in-vitro map to analyze the differentiation and polarization of human M-DM under diverse stimuli.

Monocytes and macrophages are very plastic and reprogrammable immune cells crucial for health and disease. They sense and respond to a broad range of stimuli by adopting specific differentiation programs and phenotypes. We have standardized an in-vitro model to study macrophage polarization and reprogramming, providing a valuable tool for research.

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s ...

In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

Source: Plaisance-Bonstaff, K., et al. <a href="https://app.jove.com/v/59967">Isolation, Transfection, and Culture of Primary Human Monocytes.</a> J. Vis. Exp. (2019)
This video demonstrates the priming of primary monocytes into macrophages. Primary human monocytes can be isolated and differentiated to an M1-like pro-inflammatory phenotype using the granulocyte-macrophage colony-stimulating factor (GM-CSF) or to an M2-like anti-inflammatory phenotype using the macrophage colony-stimulating factor (M-CSF).

Hodowla in vitro i różnicowanie pierwotnych monocytów do makrofagów

Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019)
This video demonstrates the ...

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

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

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Isolation, Transfection, and Culture of Primary Human Monocytes

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

Hodowla in vitro i różnicowanie pierwotnych monocytów do makrofagów