Summary
In vitro models of macrophage growth, differentiation, and function are needed to facilitate the study of their biology as important immune facilitator cells and as frequent targets of bacterial and viral infection. A simple method for the selective expansion and continuous culture of mouse macrophages from primary explant cultures of mouse embryonic tissue is described. Culture in Dulbecco modified Eagle medium (DMEM) low-glucose (1 g/L) formulation (DMEM/L) inhibited fibroblast growth. In contrast, macrophages continued to proliferate in the presence of DMEM/L when in contact with the fibroblasts. Alternating growth in high-glucose DMEM with DMEM/L produced a 1.16− to 2.1-fold increase depending on mouse strain) in the percentage of macrophages within the cell culture in comparison with culturing in DMEM with high glucose exclusively. Macrophage yields of over 1 million cells/T12.5 flask were achieved by passages 3–4, and, thereafter, declined over the next 5–10 passages. The peak percentage of macrophages within a culture varied depending on the strain of mouse (C57BL/6, CD-1, and CF-1 and two knockout C57BL/6 strains deficient in either interleukin-6 [IL-6] or granulocyte colony stimulating factor [GCSF]). The GCSF (−/−)-derived cultures had the lowest peak macrophage content (30%) and CD-1 the highest content (64.9%). The IL-6 (−/−) and CD-1 cultures appeared to spontaneously transform to create cell lines (IL6MAC and CDIMAC, respectively) that were composed of 50–75% macrophages. The macrophages were phagocytic and were positive for CD14, acetylated low-density lipoprotein receptors, and F4-80 antigen. Light and electron microscopy showed that the cultured macrophages had in vivo-like morphological features, and they could be plated to high purity by differential attachment to petri dishes in serum-free medium.
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References
Adams, D. O. Macrophages. In: Jakoby, W. B.; Pastan, H. I., ed. Methods in enzymology; cell culture. Vol. LVIII. New York: Academic Press; 1979:494–507.
Auger, M. J.; Ross, J. A. The biology of the macrophage. In: Lewis, C. E.; McGee, J. O’D., ed. The macrophage. Oxford: IRL Press; 1992:1–74.
Bennett, S.; Por, S. B.; Cooley, M. A.; Breit, S. N. In vitro replication dynamics of human culture-derived macrophages in a long term serum-free system. J. Immunol. 150:2364–2371; 1993.
Bitterman, P. B.; Wewers, M. D.; Rennard, S. I.; Adelberg, S.; Crystal, R. G. Modulation of alveolar macrophage-driven fibroblast proliferation by alternative macrophage mediators. J. Clin. Invest. 77:700–708; 1986.
Bluethmann, H.; Rothe, J.; Schultz, N.; Tkachuk, M.; Koebel, P. Establishment of the role of IL-6 and TNF receptor 1 using gene knockout mice. J. Leukoc. Biol. 56:565–570; 1994.
Cohen, A. B.; Cline, M. J. The human alveolar macrophage: isolation, cultivation in vitro, and studies of morphologic and functional characterization. J. Clin. Invest. 50:1390–1398; 1971.
Dalrymple, S. A.; Lucian, L. A.; Slattery, R.; McNeil, T.; Aud, D. M.; Fuchino, S.; Lee, F.; Murray, R. Interleukin-6-deficient mice are highly susceptible to Listeria monocytogenes infection: correlation with inefficient neutrophilia. Infect. Immun. 63:2262–2268; 1995.
Davis, S.; Gale, N. W.; Aldrich, T. H.; Maisonpierre, P. C.; Lhotak, V.; Pawson, T.; Goldfarb, M.; Yancopoulos, G. D. Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity. Science 266:816–819; 1994.
Fisher, J.; Mizrahi, T.; Schori, H.; Yoles, E.; Levkovitch-Verbin, H.; Haggiag, S.; Revel, M.; Schwartz, M. Increased post-traumatic survival of neurous in IL-6-knockout mice on a background of EAE susceptibility. J. Neuroimmunol. 119;1–9; 2001.
Freshney, R. I. Culture of animal cells. 3rd ed. New York: Wiley-Liss; 1994: 345, 107–126.
Genovesi, E. V.; Knudsen, R. C.; Gerstner, D. J.; Card, D. M.; Martins, C. L. V.; Quintero, J. C.; Whyard, T. C. In vitro induction of swine peripheral ripheral blood monocyte proliferation by the fibroblast-derived murine hematopoietic growth factor CSF-1. Vet. Immunol. Immunopathol. 23:223–244; 1989.
Goldstein, J. L.; Ho, Y. K.; Basu, S. K.; Brown, M. S. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc. Natl. Acad. Sci. USA 76:335–337; 1979.
Gonzalez-Ramos, A.; Cooper, K. D.; Hammerberg, C.. Identification of a human dermal macrophage population responsible for constitutive restraint of primary dermal fibroblast proliferation. J. Invest. Dermatol. 106:305–311; 1996.
Gualandris, A.; Annes, J. P.; Arese, M.; Noguera, I.; Jurukovski, V.; Rifkin, D. B. The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium. Mol. Biol. Cell 11:4295–4308; 2000.
Kobari, L.; Dubart, A.; Le Pesteur, F.; Vainchenker, W.; Sainteny, F. Hematopoietic-promoting activity of the murine stromal cell line MS-5 is not related to the expression of the major hematopoietic cytokines. J. Cell. Physiol. 163:295–304; 1995.
Kohchi, C.; Tanabe, Y.; Noguchi, K.; Mizuno, D.; Soma, G. Induction of differentiation in embryonic stem cells by 26-kD membrane-bound tumor necrosis factor (TNF) and 17 kD free TNF. In Vivo 10:19–27; 1996.
Kopf, M.; Baumann, H.; Freer, G., et al. Impaired immune and acute-phase response in interleukin-6-deficient mice. Nature 368:339–342; 1994.
Kramer, J.; Hegert, C.; Guan, K.; Wobus, A. M.; Muller, P. K.; Rohwedel, J. Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech. Dev. 92;193–205; 2000
Ledermann, B.; Bürki, K. Establishment of a germ-line competent C57BI/6 embryonic stem cell line. Exp. Cell. Res. 197:254–258; 1991.
Lieschke, G. J.; Grail, D.; Hodgson, G., et al. Mice lacking granulocyte-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 84:1737–1746; 1994.
Matsui, Y.; Zsebo, K.; Hogan, B. L. M. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70:841–847; 1992.
McCullough, K. C.; Schaffner, R.; Fraefel, W.; Kihm, U. The relative density of CD44-positive procine monocytic cell populations varies between isolations and upon culture and influences susceptibility to infection by African swine fever virus. Immunol. Lett. 37:83–90; 1993.
McKnight, A. J.; Macfarlane, A. J.; Dri, P.; Turley, L.; Willis, A. C.; Gordon, S. Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family. J. Biol. Chem. 271:486–489; 1996.
Naito, M.; Umeda, S.; Yamamoto, T., et al. Development, differentiation, and phenotypic heterogeneity of murine tissue macrophages. J. Leukoc. Biol. 59:133–138; 1996.
Nathan, C. F. Secretory products of macrophages. J. Clin. Invest. 79:319–326; 1987.
Naum, Y. Growth of pulmonary alveolar macrophages in vitro: responses to media conditioned by lung cell lines. Cytobios 14:211–220; 1975.
Penkowa, M.; Moos, T.; Carrasco, J.; Hadberg, H.; Molinero, A.; Bluethmann, H.; Hidalgo, J. Strongly compromised inflammatory response to brain injury in interleukin-6-deficient mice. Glia 25:343–357; 1999.
Rappolee, D. A.; Werb, Z. Macrophage-derived growth factors. In: Russell, S. W.; Gordon, S., ed. Macrophage biology and activation. Berlin: Springer-Verlag; 1992:87–140.
Roberts, R.; Gallagher, J.; Spooncer, E.; Allen, T. D.; Bloomfield, F.; Dexter, T. M. Heparin sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature 332:376–378; 1988.
Saad, A. M.; Hageltorn, M. Flow cytometric characterization of bovine blood neutrophil phagocytosis of fluorescent bacteria and zymosan particles. Acta Vet. Scand. 26:289–307; 1985.
Stanley, E. R.; Heard, P. M. Factors regulating macrophage production and growth. Purification and some properties of the colony stimulating factor from medium conditioned by mouse L cells. Biol. Chem. 252:4305–4312; 1977.
Stein, J.; Borzillo, G. V.; Rettenmier, C. W. Direct stimulation of cells expressing receptors for macrophage colony-stimulating factor (CSF-1) by plasma membrane-bound precursors of human CSF-1. Blood 76:1308–1314; 1990.
Talbot, N. C.; Garrett, W. M.; Caperna, T. J. Analysis of the expression of aquaporin-1 and-9 in pig liver tissue: comparison with rat liver tissue. Cells Tissues Organs 174:17–28; 2003.
Talbot, N. C.; Paape, M.; Worku, M. Selective and continuous culture of macrophages from adult pig blood. Vet. Immunol. Immunopathol. 64:173–190; 1998.
Talbot, N. C.; Powell, A.; Garrett, W.; Edwards, J. L.; Rexroad, C. E., Jr. Ultrastructural and karyotypic examination of in vitro produced bovine embryos developed in the sheep uterus. Tissue Cell 32:9–27; 2000.
Thomson, J. A.; Itskovitz-Eldor, J.; Shapiro, S. S.; Waknitz, M. A.; Swiergiel, J. J.; Marshall, V. S.; Jones, J. M. Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147; 1998.
Tushinski, R. J.; Oliver, I. T.; Guilbert, L. J.; Tynan, W.; Warner, J. R.; Stanley, E. R. Survival of mononuclear phagocytes depends on, a lineage-specific growth factor that the differentiated cells selectivety destroy. Cell 28:71–81; 1982.
Unanue, E. R.; Allen, P. M. The basis for the immunoregulatory role of macrophages and other accessory cells. Science 36:551–557; 1987.
Walker, W. S. Establishment of mononuclear phagocyte cell lines. J. Immunol. Methods 174:25–31; 1994.
Wright, S. D.; Ramos, R. A.; Tobias, P. S.; Ulevitch, R. J.; Mathison, J. C. CD14 a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249:1431–1433; 1990.
Wu, J.; Zhu, J. Q.; Zhu, D. X. The regulatory effects of macrophages on colony stimulating factor-1 (CSF-1) production. Int. J. Biochem. 22:513–517; 1990.
Wuu, Y. D.; Pampfer, S.; Vanderheyden, I.; Lee, K. H.; De Hertogh, R. Impact of tumor necrosis factor alpha on mouse embryonic stem cells. Biol. Reprod. 58:1416–1424; 1998.
Xu, C.; Inokuma, M. S.; Denham, J.; Golds, K.; Kundu, P.; Gold, J. D.; Carpenter, M. K. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19:971–974; 2001.
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Talbot, N.C., Paape, M., Sohn, E.J. et al. Macrophage population dynamics within fetal mouse fibroblast cultures derived from C57BL/6, CD-1, CF-1 mice and interleukin-6 and granulocyte colony stimulating factor knockout mice. In Vitro Cell.Dev.Biol.-Animal 40, 196–210 (2004). https://doi.org/10.1290/1543-706X(2004)40<196:MPDWFM>2.0.CO;2
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DOI: https://doi.org/10.1290/1543-706X(2004)40<196:MPDWFM>2.0.CO;2