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Cell Migration pp 149-165 | Cite as

In Vitro Analysis of Chemotactic Leukocyte Migration in 3D Environments

  • Michael SixtEmail author
  • Tim Lämmermann
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 769)

Abstract

Cell migration on two-dimensional (2D) substrates follows entirely different rules than cell migration in three-dimensional (3D) environments. This is especially relevant for leukocytes that are able to migrate in the absence of adhesion receptors within the confined geometry of artificial 3D extracellular matrix scaffolds and within the interstitial space in vivo. Here, we describe in detail a simple and economical protocol to visualize dendritic cell migration in 3D collagen scaffolds along chemotactic gradients. This method can be adapted to other cell types and may serve as a physiologically relevant paradigm for the directed locomotion of most amoeboid cells.

Key words

3D Interstitial migration Chemotaxis Chemokine gradient Cell motility Collagen Extracellular matrix Interstitium Connective tissue 

References

  1. 1.
    Cukierman, E., Pankov, R., Stevens, D. R., and Yamada, K. M. (2001) Taking cell-matrix adhesions to the third dimension, Science (New York, N.Y 294, 1708–1712.Google Scholar
  2. 2.
    Lämmermann, T., Bader, B. L., Monkley, S. J., Worbs, T., Wedlich-Soldner, R., Hirsch, K., Keller, M., Forster, R., Critchley, D. R., Fassler, R., and Sixt, M. (2008) Rapid leukocyte migration by integrin-independent flowing and squeezing, Nature 453, 51–55.PubMedCrossRefGoogle Scholar
  3. 3.
    Martin-Belmonte, F., and Mostov, K. (2008) Regulation of cell polarity during epithelial morphogenesis, Current opinion in cell biology 20, 227–234.PubMedCrossRefGoogle Scholar
  4. 4.
    Wolf, K., Alexander, S., Schacht, V., Coussens, L. M., von Andrian, U. H., van Rheenen, J., Deryugina, E., and Friedl, P. (2009) Collagen-based cell migration models in vitro and in vivo, Seminars in cell & developmental biology 20, 931–941.CrossRefGoogle Scholar
  5. 5.
    Wolf, K., Mazo, I., Leung, H., Engelke, K., von Andrian, U. H., Deryugina, E. I., Strongin, A. Y., Brocker, E. B., and Friedl, P. (2003) Compensation mechanism in tumor cell ­migration: mesenchymal-amoeboid transition after ­blocking of pericellular proteolysis, The Journal of cell biology 160, 267–277.PubMedCrossRefGoogle Scholar
  6. 6.
    Yamada, K. M., and Cukierman, E. (2007) Modeling tissue morphogenesis and cancer in 3D, Cell 130, 601–610.PubMedCrossRefGoogle Scholar
  7. 7.
    Reichardt, P., Gunzer, F., and Gunzer, M. (2007) Analyzing the physicodynamics of immune cells in a three-dimensional collagen matrix, Methods in molecular biology (Clifton, NJ) 380, 253–269.Google Scholar
  8. 8.
    Lämmermann, T., Renkawitz, J., Wu, X., Hirsch, K., Brakebusch, C., and Sixt, M. (2009) Cdc42-dependent leading edge coordination is essential for interstitial dendritic cell migration, Blood 113, 5703–5710.PubMedCrossRefGoogle Scholar
  9. 9.
    Lämmermann, T., and Sixt, M. (2009) Mechanical modes of “amoeboid” cell migration, Current opinion in cell biology 21, 636–644.PubMedCrossRefGoogle Scholar
  10. 10.
    Schmidt, S., and Friedl, P. (2010) Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms, Cell and tissue research 339, 83–92.PubMedCrossRefGoogle Scholar
  11. 11.
    Sabeh, F., Shimizu-Hirota, R., and Weiss, S. J. (2009) Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited, The Journal of cell biology 185, 11–19.PubMedCrossRefGoogle Scholar
  12. 12.
    Peters, N. C., Egen, J. G., Secundino, N., Debrabant, A., Kimblin, N., Kamhawi, S., Lawyer, P., Fay, M. P., Germain, R. N., and Sacks, D. (2008) In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies, Science (New York, N.Y 321, 970–974.Google Scholar
  13. 13.
    Pflicke, H., and Sixt, M. (2009) Preformed portals facilitate dendritic cell entry into afferent lymphatic vessels, The Journal of experimental medicine 206, 2925–2935.PubMedCrossRefGoogle Scholar
  14. 14.
    Shields, J. D., Fleury, M. E., Yong, C., Tomei, A. A., Randolph, G. J., and Swartz, M. A. (2007) Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling, Cancer cell 11, 526–538.PubMedCrossRefGoogle Scholar
  15. 15.
    Quast, T., Tappertzhofen, B., Schild, C., Grell, J., Czeloth, N., Forster, R., Alon, R., Fraemohs, L., Dreck, K., Weber, C., Lämmermann, T., Sixt, M., and Kolanus, W. (2009) Cytohesin-1 controls the activation of RhoA and modulates integrin-dependent adhesion and migration of dendritic cells, Blood 113, 5801–5810.PubMedCrossRefGoogle Scholar
  16. 16.
    Brightman, A. O., Rajwa, B. P., Sturgis, J. E., McCallister, M. E., Robinson, J. P., and Voytik-Harbin, S. L. (2000) Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro, Biopolymers 54, 222–234.PubMedCrossRefGoogle Scholar
  17. 17.
    Bonvin, C., Overney, J., Shieh, A. C., Dixon, J. B., and Swartz, M. A. (2010) A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: development, characterization, and applications, Biotechnology and bioengineering 105, 982–991.PubMedGoogle Scholar
  18. 18.
    Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., and Schuler, G. (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow, Journal of immunological methods 223, 77–92.PubMedCrossRefGoogle Scholar
  19. 19.
    Menges, M., Rossner, S., Voigtlander, C., Schindler, H., Kukutsch, N. A., Bogdan, C., Erb, K., Schuler, G., and Lutz, M. B. (2002) Repetitive injections of dendritic cells matured with tumor necrosis factor alpha induce antigen-specific protection of mice from autoimmunity, The Journal of experimental medicine 195, 15–21.PubMedCrossRefGoogle Scholar
  20. 20.
    Huang, J. H., Cardenas-Navia, L. I., Caldwell, C. C., Plumb, T. J., Radu, C. G., Rocha, P. N., Wilder, T., Bromberg, J. S., Cronstein, B. N., Sitkovsky, M., Dewhirst, M. W., and Dustin, M. L. (2007) Require-ments for T lymphocyte migration in explanted lymph nodes, J Immunol 178, 7747–7755.PubMedGoogle Scholar
  21. 21.
    Haessler, U., Kalinin, Y., Swartz, M. A., and Wu, M. (2009) An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies, Biomedical microdevices 11, 827–835.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Institute of Science and Technology AustriaKlosterneuburgAustria

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