Advertisement

Microglia Cell Culture: A Primer for the Novice

  • Anke Witting
  • Thomas MöllerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 758)

Abstract

Microglial cells are the resident immune cells of the central nervous system. Progress in the recent decade has clearly established that microglial cells participate or even actively drive neurological disease. Much of our current knowledge has been generated by investigating microglial cells in cell culture. The aim of this chapter is to give the uninitiated a basic and adaptable protocol for the culturing of microglial cells. We discuss the challenges of microglial cell culture and provide a collection of tips which reflect our 25+ years of collective experience.

Key words

Microglia Cell culture Medium Serum Growth factor M-CSF GM-CSF Transfection Endotoxin Cell yield 

References

  1. 1.
    Ransohoff, R. M., and Perry, V. H. (2009) Microglial physiology: unique stimuli, specialized responses, Annu Rev Immunol 27, 119–145.PubMedCrossRefGoogle Scholar
  2. 2.
    Hanisch, U. K., and Kettenmann, H. (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain, Nature Neuroscience 10, 1387–1394.PubMedCrossRefGoogle Scholar
  3. 3.
    Biber, K., Neumann, H., Inoue, K., and Boddeke, H. W. (2007) Neuronal ‘On’ and ‘Off’ signals control microglia, Trends Neurosci 30, 596–602.PubMedCrossRefGoogle Scholar
  4. 4.
    Garden, G. A., and Möller, T. (2006) Microglia biology in health and disease, J Neuroimmune Pharmacol 1, 127–137.PubMedCrossRefGoogle Scholar
  5. 5.
    Schwartz, M., Butovsky, O., Bruck, W., and Hanisch, U. K. (2006) Microglial phenotype: is the commitment reversible?, Trends Neurosci 29, 68–74.PubMedCrossRefGoogle Scholar
  6. 6.
    Chan, W. Y., Kohsaka, S., and Rezaie, P. (2007) The origin and cell lineage of microglia: new concepts, Brain Res Rev 53, 344–354.PubMedCrossRefGoogle Scholar
  7. 7.
    Graeber, M. B., and Streit, W. J. (2010) Microglia: biology and pathology, Acta Neuropathol 119, 89–105.PubMedCrossRefGoogle Scholar
  8. 8.
    van Rossum, D., and Hanisch, U. K. (2004) Microglia, Metab Brain Dis 19, 393–411.PubMedCrossRefGoogle Scholar
  9. 9.
    Sugama, S., Takenouchi, T., Cho, B. P., Joh, T. H., Hashimoto, M., and Kitani, H. (2009) Possible roles of microglial cells for neurotoxicity in clinical neurodegenerative diseases and experimental animal models, Inflamm Allergy Drug Targets 8, 277–284.PubMedCrossRefGoogle Scholar
  10. 10.
    Carson, M. J. (2002) Microglia as liaisons between the immune and central nervous systems: functional implications for multiple sclerosis, Glia 40, 218–231.PubMedCrossRefGoogle Scholar
  11. 11.
    Garden, G. A. (2002) Microglia in human immunodeficiency virus-associated neurodegeneration, Glia 40, 240–251.PubMedCrossRefGoogle Scholar
  12. 12.
    Appel, S. H., Beers, D. R., and Henkel, J. S. (2010) T cell-microglial dialogue in Parkinson’s disease and amyotrophic lateral sclerosis: are we listening?, Trends Immunol 31, 7–17.PubMedCrossRefGoogle Scholar
  13. 13.
    Raibon, E., Todd, L. M., and Moller, T. (2008) Glial Cells in ALS: The Missing Link?, Phys Med Rehabil Clin N Am 19, 441–459.PubMedCrossRefGoogle Scholar
  14. 14.
    Streit, W. J. (2004) Microglia and Alzheimer’s disease pathogenesis, J Neurosci Res 77, 1–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Kim, Y. S., and Joh, T. H. (2006) Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson’s disease, Experimental & molecular medicine 38, 333–347.CrossRefGoogle Scholar
  16. 16.
    Giulian, D., and Baker, T. J. (1986) Characterization of ameboid microglia isolated from developing mammalian brain, J Neurosci 6, 2163–2178.PubMedGoogle Scholar
  17. 17.
    Schmid, C. D., Melchior, B., Masek, K., Puntambekar, S. S., Danielson, P. E., Lo, D. D., Sutcliffe, J. G., and Carson, M. J. (2009) Differential gene expression in LPS/IFNgamma activated microglia and macrophages: in vitro versus in vivo, J Neurochem 109 Suppl 1, 117–125.PubMedCrossRefGoogle Scholar
  18. 18.
    Carson, M. J., Crane, J., and Xie, A. X. (2008) Modeling CNS microglia: the quest to identify predictive models, Drug Discov Today Dis Models 5, 19–25.PubMedCrossRefGoogle Scholar
  19. 19.
    Nimmerjahn, A., Kirchhoff, F., and Helmchen, F. (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo, Science 308, 1314–1318.PubMedCrossRefGoogle Scholar
  20. 20.
    Davalos, D., Grutzendler, J., Yang, G., Kim, J. V., Zuo, Y., Jung, S., Littman, D. R., Dustin, M. L., and Gan, W. B. (2005) ATP mediates rapid microglial response to local brain injury in vivo, Nature neuroscience 8, 752–758.PubMedCrossRefGoogle Scholar
  21. 21.
    Fuhrmann, M., Bittner, T., Jung, C. K., Burgold, S., Page, R. M., Mitteregger, G., Haass, C., LaFerla, F. M., Kretzschmar, H., and Herms, J. (2010) Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease, Nature neuroscience 13, 411–413.PubMedCrossRefGoogle Scholar
  22. 22.
    Boillee, S., Yamanaka, K., Lobsiger, C. S., Copeland, N. G., Jenkins, N. A., Kassiotis, G., Kollias, G., and Cleveland, D. W. (2006) Onset and Progression in Inherited ALS Determined by Motor Neurons and Microglia, Science 312, 1389–1392.PubMedCrossRefGoogle Scholar
  23. 23.
    Moussaud, S., and Draheim, H. J. (2010) A new method to isolate microglia from adult mice and culture them for an extended period of time, J Neurosci Methods 187, 243–253.PubMedCrossRefGoogle Scholar
  24. 24.
    Marshall, G. P., 2nd, Demir, M., Steindler, D. A., and Laywell, E. D. (2008) Subventricular zone microglia possess a unique capacity for massive in vitro expansion, Glia 56, 1799–1808.PubMedCrossRefGoogle Scholar
  25. 25.
    Napoli, I., Kierdorf, K., and Neumann, H. (2009) Microglial precursors derived from mouse embryonic stem cells, Glia 57, 1660–1671.PubMedCrossRefGoogle Scholar
  26. 26.
    McLarnon, J. G., Xu, R., Lee, Y. B., and Kim, S. U. (1997) Ion channels of human microglia in culture, Neuroscience 78, 1217–1228.PubMedCrossRefGoogle Scholar
  27. 27.
    Narayan, P. J., Gibbons, H. M., Mee, E. W., Faull, R. L., and Dragunow, M. (2007) High throughput quantification of cells with complex morphology in mixed cultures, J Neurosci Methods 164, 339–349.PubMedCrossRefGoogle Scholar
  28. 28.
    De Groot, C. J., Montagne, L., Janssen, I., Ravid, R., Van Der Valk, P., and Veerhuis, R. (2000) Isolation and characterization of adult microglial cells and oligodendrocytes derived from postmortem human brain tissue, Brain Res Brain Res Protoc 5, 85–94.PubMedCrossRefGoogle Scholar
  29. 29.
    Broderick, C., Duncan, L., Taylor, N., and Dick, A. D. (2000) IFN-gamma and LPS-mediated IL-10-dependent suppression of retinal microglial activation, Invest Ophthalmol Vis Sci 41, 2613–2622.PubMedGoogle Scholar
  30. 30.
    Tambuyzer, B. R., and Nouwen, E. J. (2005) Inhibition of microglia multinucleated giant cell formation and induction of differentiation by GM-CSF using a porcine in vitro model, Cytokine 31, 270–279.PubMedCrossRefGoogle Scholar
  31. 31.
    Houalla, T., and Levine, R. L. (2003) The isolation and culture of microglia-like cells from the goldfish brain, J Neurosci Methods 131, 121–131.PubMedCrossRefGoogle Scholar
  32. 32.
    Cole, R., and de Velis, J. (2001) Preparation of astrocyte, oligodendrocyte, and microglia cultures from primary rat cerebral cultures, In Protocols for Neural Cell Culture (Fedoroff, S., and Richardson, A., Eds.).Google Scholar
  33. 33.
    Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. C., Trent, J. M., Staudt, L. M., Hudson, J., Jr., Boguski, M. S., Lashkari, D., Shalon, D., Botstein, D., and Brown, P. O. (1999) The transcriptional program in the response of human fibroblasts to serum, Science 283, 83–87.PubMedCrossRefGoogle Scholar
  34. 34.
    Möller, T., Weinstein, J. R., and Hanisch, U. K. (2006) Activation of microglial cells by thrombin: past, present, and future, Seminars in thrombosis and hemostasis 32 Suppl 1, 69–76.PubMedCrossRefGoogle Scholar
  35. 35.
    Möller, T., Kann, O., Prinz, M., Kirchhoff, F., Verkhratsky, A., and Kettenmann, H. (1997) Endothelin-induced calcium signaling in ­cultured mouse microglial cells is mediated through ETB receptors, Neuroreport 8, 2127–2131.PubMedCrossRefGoogle Scholar
  36. 36.
    Möller, T., Contos, J. J., Musante, D. B., Chun, J., and Ransom, B. R. (2001) Expression and function of lysophosphatidic acid receptors in cultured rodent microglial cells, J Biol Chem 276, 25946–25952.PubMedCrossRefGoogle Scholar
  37. 37.
    Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison, J. C., Tobias, P. S., and Ulevitch, R. J. (1990) Structure and function of lipopolysaccharide binding protein, Science 249, 1429–1431.PubMedCrossRefGoogle Scholar
  38. 38.
    Wang, R. C., and Levine, B. (2010) Autophagy in cellular growth control, FEBS Lett 584, 1417–1426.PubMedCrossRefGoogle Scholar
  39. 39.
    Janeway, C. A., Travers, P., and Walport, M. (2007) Immunobiology: The Immune System in Health and Disease, Garland Science, Taylor and Francis Group, New York and London.Google Scholar
  40. 40.
    Dixon, D. R., and Darveau, R. P. (2005) Lipopolysaccharide heterogeneity: innate host responses to bacterial modification of lipid a structure, J Dent Res 84, 584–595.PubMedCrossRefGoogle Scholar
  41. 41.
    Westphal, O., Luderitz, O., Rietschel, E. T., and Galanos, C. (1981) Bacterial lipopolysaccharide and its lipid A component: some ­historical and some current aspects, Biochem Soc Trans 9, 191–195.PubMedGoogle Scholar
  42. 42.
    Palsson-McDermott, E. M., and O’Neill, L. A. (2004) Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4, Immunology 113, 153–162.PubMedCrossRefGoogle Scholar
  43. 43.
    Weinstein, J. R., Swarts, S., Bishop, C., Hanisch, U. K., and Moller, T. (2008) Lipopolysaccharide is a frequent and significant contaminant in microglia-activating factors, Glia 56, 16–26.PubMedCrossRefGoogle Scholar
  44. 44.
    Re, F., Belyanskaya, S. L., Riese, R. J., Cipriani, B., Fischer, F. R., Granucci, F., Ricciardi-Castagnoli, P., Brosnan, C., Stern, L. J., Strominger, J. L., and Santambrogio, L. (2002) Granulocyte-macrophage colony-stimulating factor induces an expression program in neonatal microglia that primes them for antigen presentation, J Immunol 169, 2264–2273.PubMedGoogle Scholar
  45. 45.
    Vidyadaran, S., Ooi, Y. Y., Subramaiam, H., Badiei, A., Abdullah, M., Ramasamy, R., and Seow, H. F. (2009) Effects of macrophage colony-stimulating factor on microglial responses to lipopolysaccharide and beta amyloid, Cell Immunol 259, 105–110.PubMedCrossRefGoogle Scholar
  46. 46.
    Esen, N., and Kielian, T. (2007) Effects of low dose GM-CSF on microglial inflammatory profiles to diverse pathogen-associated molecular patterns (PAMPs), J Neuroinflammation 4, 10.PubMedCrossRefGoogle Scholar
  47. 47.
    Hamilton, J. A. (2008) Colony-stimulating factors in inflammation and autoimmunity, Nat Rev Immunol 8, 533–544.PubMedCrossRefGoogle Scholar
  48. 48.
    Barreda, D. R., Hanington, P. C., and Belosevic, M. (2004) Regulation of myeloid development and function by colony stimulating factors, Dev Comp Immunol 28, 509–554.PubMedCrossRefGoogle Scholar
  49. 49.
    Martinez, F. O., Helming, L., and Gordon, S. (2009) Alternative activation of macrophages: an immunologic functional perspective, Annu Rev Immunol 27, 451–483.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Neurology and Center for Neurogenetics and NeurotherapeuticsUniversity of WashingtonSeattleUSA

Personalised recommendations