JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Immunology and Infection

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

Published: June 30th, 2016

DOI:

10.3791/54244

1Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, 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.

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.

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 tissue....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Leukapheresis was carried out under a human subject's research protocol approved by a National Institutes of Health institutional review board.

1. Isolation and Cryopreservation of Monocytes

  1. 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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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-.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Dey, A., Allen, J., Hankey-Giblin, P. A. Ontogeny and polarization of macrophages in inflammation: blood monocytes versus tissue macrophages. Front. Immunol. 5, 683 (2014).
  2. Waldo, S. W., et al. Heterogeneity of human macrophages in culture and in atherosclerotic plaques. Am. J. Pathol. 172, 1112-1126 (2008).
  3. Akagawa, K. S. Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages. Int. J. Hematol. 76, 27-34 (2002).
  4. Akagawa, K. S., et al. Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages. Respirology. 11 Suppl, S32-S36 (2006).
  5. Fleetwood, A. J., Lawrence, T., Hamilton, J. A., Cook, A. D. 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. J. Immunol. 178, 5245-5252 (2007).
  6. Lacey, D. C., et al. Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models. J. Immunol. 188, 5752-5765 (2012).
  7. Strasser, E. F., Eckstein, R. Optimization of leukocyte collection and monocyte isolation for dendritic cell culture. Transfus. Med. Rev. 24, 130-139 (2010).
  8. Kim, S., et al. Monocyte enrichment from leukapheresis products by using the Elutra cell separator. Transfusion (Paris). 47, 2290-2296 (2007).
  9. Strober, W. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. Appendix 3 (Appendix 3B), (2001).
  10. Anzinger, J. J., et al. Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis. Arterioscler. Thromb. Vasc. Biol. 30, 2022-2031 (2010).
  11. Zhao, B., et al. Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF). J. Biol. Chem. 281, 15757-15762 (2006).
  12. Freeman, S. R., et al. ABCG1-mediated generation of extracellular cholesterol microdomains. J. Lipid Res. 55, 115-127 (2014).
  13. Kruth, H. S. Receptor-independent fluid-phase pinocytosis mechanisms for induction of foam cell formation with native low-density lipoprotein particles. Curr. Opin. Lipidol. 22, 386-393 (2011).
  14. Jin, X., et al. ABCA1 contributes to macrophage deposition of extracellular cholesterol. J. Lipid Res. 56, 1720-1726 (2015).
  15. Ong, D. S., et al. Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport. J. Lipid Res. 51, 2303-2313 (2010).
  16. Hashimoto, S., Yamada, M., Motoyoshi, K., Akagawa, K. S. Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10. Blood. 89, 315-321 (1997).
  17. Hashimoto, S. I., Komuro, I., Yamada, M., Akagawa, K. S. 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. J. Immunol. 167, 3619-3625 (2001).
  18. Lund, P. K., Joo, G. B., Westvik, A. B., Ovstebo, R., Kierulf, P. Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years' experience. Scand. J. Clin. Lab. Invest. 60, 357-365 (2000).
  19. Weiner, R. S., Normann, S. J. Functional integrity of cryopreserved human monocytes. J. Natl. Cancer Inst. 66, 255-260 (1981).
  20. Hansen, J. B., et al. Retention of phagocytic functions in cryopreserved human monocytes. J. Leukoc. Biol. 57, 235-241 (1995).
  21. Seager Danciger, J., et al. Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation. J. Immunol. Methods. 288, 123-134 (2004).
  22. Jin, X., Xu, Q., Champion, K., Kruth, H. S. Endotoxin contamination of apolipoprotein A-I: effect on macrophage proliferation--a cautionary tale. Atherosclerosis. 240, 121-124 (2015).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved