Biomedical Microdevices

, Volume 11, Issue 4, pp 827–835 | Cite as

An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies

  • Ulrike Haessler
  • Yevgeniy Kalinin
  • Melody A. Swartz
  • Mingming Wu


The current state-of-art in 3D microfluidic chemotaxis device (μFCD) is limited by the inherent coupling of the fluid flow and chemical concentration gradients. Here, we present an agarose-based 3D μFCD that decouples these two important parameters, in that the flow control channels are separated from the cell compartment by an agarose gel wall. This decoupling is enabled by the transport property of the agarose gel, which—in contrast to the conventional microfabrication material such as polydimethylsiloxane (PDMS)—provides an adequate physical barrier for convective fluid flow while at the same time readily allowing protein diffusion. We demonstrate that in this device, a gradient can be pre-established in an agarose layer above the cell compartment (a gradient buffer) before adding the 3D cell-containing matrix, and the dextran (10 kDa) concentration gradients can be re-established within 10 min across the cell-containing matrix and remain stable indefinitely. We successfully quantified the chemotactic response of murine dendritic cells to a gradient of CCL19, an 8.8 kDa lymphoid chemokine, within a type I collagen matrix. This model system is easy to set up, highly reproducible, and will benefit research on 3D chemoinvasion studies, for example with cancer cells or immune cells. Because of its gradient buffering capacity, it is particularly suitable for studying rapidly migrating cells like mature dendritic cells and neutrophils.


Microfluidics Cell motility Chemotaxis Extracellular matrix 



MW would like to thank all members of the Swartz lab during her half year visit there, MW and YK acknowledge the very helpful technical support from Andrew Darling and Nak Won Choi. MW and YK were supported by funds from the National Science Foundation (CBET-0619626) and by grants from the Nanobiotechnology Center (NBTC), an STC Program of the National Science Foundation under Agreement No. ECS-9876771. MAS and UH were supported by the Swiss National Science Foundation (107602 and 310010).

Supplementary material

10544_2009_9299_MOESM1_ESM.avi (89.1 mb)
SM1 Movie of murine dentritic cells migrating in a 3D collagen matrix in the absence of a chemokine gradient. Two hours total were recorded. The scale bar, 50 μm. (AVI 91259 kb)
10544_2009_9299_MOESM2_ESM.avi (89.9 mb)
SM2 Movie of murine dentritic cells migrating in a 3D collagen matrix and in the presence of a CCL19 chemokine gradient of 0.11 nM/μm. Two hours total were recorded. The scale bar, 50 μm. (AVI 92026 kb)


  1. V.V. Abhyankar, M.A. Lokuta, A. Huttenlocher, D.J. Beebe, Characterization of a membrane-based gradient generator for use in cell-signaling studies. Lab Chip 6(3), 389–393 (2006). doi: 10.1039/b514133h CrossRefGoogle Scholar
  2. V.V. Abhyankar, M.W. Toepke, C.L. Cortesio, M.A. Lokuta, A. Huttenlocher, D.J. Beebe, A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment. Lab Chip 8(9), 1507–1515 (2008). doi: 10.1039/b803533d CrossRefGoogle Scholar
  3. J. Behnsen, P. Narang, M. Hasenberg, F. Gunzer, U. Bilitewski, N. Klippel, M. Rohde, M. Brock, A.A. Brakhage, M. Gunzer, Environmental dimensionality controls the interaction of phagocytes with the pathogenic fungi Aspergillus fumigatus and Candida albicans. PLoS Pathog 3(2), e13 (2007). doi: 10.1371/journal.ppat.0030013 CrossRefGoogle Scholar
  4. S. Boyden, The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exp. Med. 115, 453–466 (1962). doi: 10.1084/jem.115.3.453 CrossRefGoogle Scholar
  5. S.Y. Cheng, S. Heilman, M. Wasserman, S. Archer, M.L. Shuler, M. Wu, A hydrogel-based microfluidic device for the studies of directed cell migration. Lab Chip 7(6), 763–769 (2007). doi: 10.1039/b618463d CrossRefGoogle Scholar
  6. E. Cukierman, R. Pankov, D.R. Stevens, K.M. Yamada, Taking cell-matrix adhesions to the third dimension. Science 294(5547), 1708–1712 (2001). doi: 10.1126/science.1064829 CrossRefGoogle Scholar
  7. J. Diao, L. Young, S. Kim, E.A. Fogarty, S.M. Heilman, P. Zhou, M.L. Shuler, M. Wu, M.P. DeLisa, A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis. Lab Chip 6(3), 381–388 (2006). doi: 10.1039/b511958h CrossRefGoogle Scholar
  8. C.W. Frevert, G. Boggy, T.M. Keenan, A. Folch, Measurement of cell migration in response to an evolving radial chemokine gradient triggered by a microvalve. Lab Chip 6(7), 849–856 (2006). doi: 10.1039/b515560f CrossRefGoogle Scholar
  9. P. Friedl, K. Maaser, C.E. Klein, B. Niggemann, G. Krohne, K.S. Zanker, Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44. Cancer Res. 57(10), 2061–2070 (1997)Google Scholar
  10. L.G. Griffith, M.A. Swartz, Capturing complex 3D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 7(3), 211–224 (2006). doi: 10.1038/nrm1858 CrossRefGoogle Scholar
  11. M. Gunzer, E. Kampgen, E.B. Brocker, K.S. Zanker, P. Friedl, Migration of dendritic cells in 3D-collagen lattices. Visualisation of dynamic interactions with the substratum and the distribution of surface structures via a novel confocal reflection imaging technique. Adv. Exp. Med. Biol. 417, 97–103 (1997)Google Scholar
  12. M. Gunzer, P. Friedl, B. Niggemann, E.B. Broker, E. Kampgen, K.S. Zanker, Migration of dendritic cells within 3-D collagen lattices is dependent on tissue origin, state of maturation, and matrix structure and is maintained by proinflammatory cytokines. J. Leukoc. Biol. 67(5), 622–629 (2000).Google Scholar
  13. K. Inaba, M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R.M. Steinman, Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176(6), 1693–1702 (1992). doi: 10.1084/jem.176.6.1693 CrossRefGoogle Scholar
  14. D. Irimia, S.Y. Liu, W.G. Tharp, A. Samadani, M. Toner, M.C. Poznansky, Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients. Lab Chip 6(2), 191–198 (2006). doi: 10.1039/b511877h CrossRefGoogle Scholar
  15. D. Irimia, G. Charras, N. Agrawal, T. Mitchison, M. Toner, Polar stimulation and constrained cell migration in microfluidic channels. Lab Chip 7(12), 1783–1790 (2007). doi: 10.1039/b710524j CrossRefGoogle Scholar
  16. X. Jiang, Q. Xu, S.K. Dertinger, A.D. Stroock, T.M. Fu, G.M. Whitesides, A general method for patterning gradients of biomolecules on surfaces using microfluidic networks. Anal. Chem. 77(8), 2338–2347 (2005). doi: 10.1021/ac048440m CrossRefGoogle Scholar
  17. L. Lebrun, G.A. Junter, Diffusion of sucrose and dextran through agar gel membranes. Enzyme Microb. Technol. 15(12), 1057–1062 (1993). doi: 10.1016/0141-0229(93)90054-6 CrossRefGoogle Scholar
  18. N. Li Jeon, H. Baskaran, S.K. Dertinger, G.M. Whitesides, L. Van de Water, M. Toner, Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device. Nat. Biotechnol. 20(8), 826–830 (2002)Google Scholar
  19. B. Mosadegh, C. Huang, J.W. Park, H.S. Shin, B.G. Chung, S.K. Hwang, K.H. Lee, H.J. Kim, J. Brody, N.L. Jeon, Generation of stable complex gradients across two-dimensional surfaces and three-dimensional gels. Langmuir 23(22), 10910–10912 (2007). doi: 10.1021/la7026835 CrossRefGoogle Scholar
  20. D.D. Patel, W. Koopmann, T. Imai, L.P. Whichard, O. Yoshie, M.S. Krangel, Chemokines have diverse abilities to form solid phase gradients. Clin. Immunol. 99(1), 43–52 (2001). doi: 10.1006/clim.2000.4997 CrossRefGoogle Scholar
  21. J.A. Pedersen, M.A. Swartz, Mechanobiology in the third dimension. Ann. Biomed. Eng. 33(11), 1469–1490 (2005). doi: 10.1007/s10439-005-8159-4 CrossRefGoogle Scholar
  22. J.A. Pedersen, F. Boschetti, M.A. Swartz, Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix. J. Biomech. 40(7), 1484–1492 (2007). doi: 10.1016/j.jbiomech.2006.06.023 CrossRefGoogle Scholar
  23. C.E. Semino, R.D. Kamm, D.A. Lauffenburger, Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow. Exp. Cell Res. 312(3), 289–298 (2006)Google Scholar
  24. A. Shamloo, N. Ma, M.M. Poo, L.L. Sohn, S.C. Heilshorn, Endothelial cell polarization and chemotaxis in a microfluidic device. Lab Chip 8(8), 1292–1299 (2008). doi: 10.1039/b719788h CrossRefGoogle Scholar
  25. J.D. Shields, M.E. Fleury, C. Yong, A.A. Tomei, G.J. Randolph, M.A. Swartz, Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling. Cancer Cell 11(6), 526–538 (2007). doi: 10.1016/j.ccr.2007.04.020 CrossRefGoogle Scholar
  26. K. Sun, Z. Wang, X. Jiang, Modular microfluidics for gradient generation. Lab Chip, 2008Google Scholar
  27. V. Vickerman, J. Blundo, S. Chung, R. Kamm, Design, fabrication and implementation of a novel multi-parameter control microfluidic platform for three-dimensional cell culture and real-time imaging. Lab Chip 8(9), 1468–1477 (2008). doi: 10.1039/b802395f CrossRefGoogle Scholar
  28. K. Wolf, P. Friedl, Mapping proteolytic cancer cell-extracellular matrix interfaces. Clin Exp Metastasis, 2008Google Scholar
  29. K. Wolf, Y.I. Wu, Y. Liu, J. Geiger, E. Tam, C. Overall, M.S. Stack, P. Friedl, Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nat. Cell Biol. 9(8), 893–904 (2007). doi: 10.1038/ncb1616 CrossRefGoogle Scholar
  30. M.H. Zaman, L.M. Trapani, A. Siemeski, D. Mackellar, H. Gong, R.D. Kamm, A. Wells, D.A. Lauffenburger, P. Matsudaira, Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proc. Natl. Acad. Sci. USA 103(29), 10889–10894 (2006). doi: 10.1073/pnas.0604460103 CrossRefGoogle Scholar
  31. S.H. Zigmond, Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. J. Cell Biol. 75(2 Pt 1), 606–616 (1977). doi: 10.1083/jcb.75.2.606 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Ulrike Haessler
    • 1
  • Yevgeniy Kalinin
    • 3
  • Melody A. Swartz
    • 1
    • 2
  • Mingming Wu
    • 3
    • 4
  1. 1.Institute of Bioengineering, School of Life SciencesÉcole Polytechnique Fédérale de Lausanne (EPFL)LausanneSwitzerland
  2. 2.Institute of Chemical Sciences and Engineering, School of Basic SciencesÉcole Polytechnique Fédérale de Lausanne (EPFL)LausanneSwitzerland
  3. 3.School of Chemical and Biomolecular EngineeringCornell UniversityIthacaUSA
  4. 4.Sibley School of Mechanical and Aerospace EngineeringCornell UniversityIthacaUSA

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