Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Reconfigurable open microfluidics for studying the spatiotemporal dynamics of paracrine signalling

Abstract

The study of intercellular signalling networks requires organotypic microscale systems that facilitate the culture, conditioning and manipulation of cells. Here, we describe a reconfigurable microfluidic cell-culture system that facilitates the assembly of three-dimensional tissue models by stacking layers that contain preconditioned microenvironments. By using principles of open and suspended microfluidics, the Stacks system is easily assembled or disassembled to provide spatial and temporal manoeuvrability in two-dimensional and three-dimensional assays of multiple cell types, enabling the modelling of sequential paracrine-signalling events, such as tumour-cell-mediated differentiation of macrophages and macrophage-facilitated angiogenesis. We used Stacks to recapitulate the in vivo observation that different prostate cancer tissues polarize macrophages with distinct gene-expression profiles of pro-inflammatory and anti-inflammatory cytokines. Stacks also enabled us to show that these two types of macrophages signal distinctly to endothelial cells, leading to blood vessels with different morphologies. Our proof-of-concept experiments exemplify how Stacks can efficiently model multicellular interactions and highlight the importance of spatiotemporal specificity in intercellular signalling.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Schematic and concept of Stacks, a reconfigurable multilayer suspended microfluidic system.
Fig. 2: Principles of suspended microfluidics enable layers to be stacked without bonding and without leaking outside the channels.
Fig. 3: Reconfigurable Stacks system enables addition and removal of macrophages at different stages to study chronological effects in cell signalling.
Fig. 4: A sequential Stacks assay was performed to achieve organotypic differentiation of TAMs in 2D culture.
Fig. 5: A sequential Stacks assay was performed to achieve organotypic differentiation of TAMs in 3D culture.
Fig. 6: Selected transcriptional profile of od-TAMs generated from culture with primary prostate cancer punch-biopsy samples.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information. Source data for the primary-sample figures are available in Figshare with the identifier https://figshare.com/s/c237024425f6e06e8fcc.

References

  1. 1.

    Chanmee, T., Ontong, P., Konno, K. & Itano, N. Tumor-associated macrophages as major players in the tumor microenvironment. Cancers 6, 1670–1690 (2014).

    CAS  Article  Google Scholar 

  2. 2.

    Qian, B.-Z. & Pollard, J. W. Macrophage diversity enhances tumor progression and metastasis. Cell 141, 39–51 (2010).

    CAS  Article  Google Scholar 

  3. 3.

    Van Ginderachter, J. A. et al. Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion. Immunobiology 211, 487–501 (2006).

    Article  Google Scholar 

  4. 4.

    Mantovani, A. & Allavena, P. The interaction of anticancer therapies with tumor-associated macrophages. J. Exp. Med. 212, 435–445 (2015).

    CAS  Article  Google Scholar 

  5. 5.

    Ostrand-Rosenberg, S. Immune surveillance: a balance between protumor and antitumor immunity. Curr. Opin. Genet. Dev. 18, 11–18 (2008).

    CAS  Article  Google Scholar 

  6. 6.

    Pollard, J. W. Tumour-educated macrophages promote tumour progression and metastasis. Nat. Rev. Cancer 4, 71–78 (2004).

    CAS  Article  Google Scholar 

  7. 7.

    Lee, G. T. et al. Macrophages induce neuroendocrine differentiation of prostate cancer cells via BMP6-IL6 Loop. Prostate 71, 1525–1537 (2011).

    CAS  PubMed  Google Scholar 

  8. 8.

    Lissbrant, I. F. et al. Tumor associated macrophages in human prostate cancer: relation to clinicopathological variables and survival. Int. J. Oncol. 17, 445–451 (2000).

    CAS  PubMed  Google Scholar 

  9. 9.

    Mestas, J. & Hughes, C. C. W. Of mice and not men: differences between mouse and human immunology. J. Immunol. 172, 2731–2738 (2004).

    CAS  Article  Google Scholar 

  10. 10.

    Pompano, R. R., Li, H.-W. & Ismagilov, R. F. Rate of mixing controls rate and outcome of autocatalytic processes: theory and microfluidic experiments with chemical reactions and blood coagulation. Biophys. J. 95, 1531–1543 (2008).

    CAS  Article  Google Scholar 

  11. 11.

    Mantovani, A., Schioppa, T., Porta, C., Allavena, P. & Sica, A. Role of tumor-associated macrophages in tumor progression and invasion. Cancer Metast. Rev. 25, 315–322 (2006).

    Article  Google Scholar 

  12. 12.

    Mantovani, A., Sozzani, S., Locati, M., Allavena, P. & Sica, A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23, 549–555 (2002).

    CAS  Article  Google Scholar 

  13. 13.

    Martinez, F. O. Macrophage activation and polarization. Front. Biosci. 13, 453–461 (2008).

    CAS  Article  Google Scholar 

  14. 14.

    Bowdish, D. M. E. in Encyclopedia of Immunobiology (Ratcliffe, M.) 289–292 (Acad. Press, 2016).

  15. 15.

    Martinez, F. O. & Gordon, S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 6, 13 (2014).

    Article  Google Scholar 

  16. 16.

    Rőszer, T. Understanding the mysterious M2 macrophage through activation markers and effector mechanisms. Mediat. Inflamm. 2015, 816460 (2015).

    Article  Google Scholar 

  17. 17.

    Nahrendorf, M. & Swirski, F. K. Abandoning M1/M2 for a network model of macrophage function. Circ. Res. 119, 414–417 (2016).

    CAS  Article  Google Scholar 

  18. 18.

    Perestrelo, A. R., Águas, A. C. P., Rainer, A. & Forte, G. Microfluidic organ/body-on-a-chip devices at the convergence of biology and microengineering. Sensors 15, 31142–31170 (2015).

    Article  Google Scholar 

  19. 19.

    Bhatia, S. N. & Ingber, D. E. Microfluidic organs-on-chips. Nat. Biotechnol. 32, 760–772 (2014).

    CAS  Article  Google Scholar 

  20. 20.

    Curtis, A. et al. Patterning of sharp cellular interfaces with a reconfigurable elastic substrate. Integr. Biol. 9, 50–57 (2017).

    CAS  Article  Google Scholar 

  21. 21.

    Ehsan, S. M., Welch-Reardon, K. M., Waterman, M. L., Hughes, C. C. W. & George, S. C. A three-dimensional in vitro model of tumor cell intravasation. Integr. Biol. 6, 603–610 (2014).

    CAS  Article  Google Scholar 

  22. 22.

    Gracz, A. D. et al. A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis. Nat. Cell Biol. 17, 340–349 (2015).

    CAS  Article  Google Scholar 

  23. 23.

    Yates, C. et al. Novel three-dimensional organotypic liver bioreactor to directly visualize early events in metastatic progression. Adv. Cancer Res. 97, 225–246 (2007).

    Article  Google Scholar 

  24. 24.

    Berthier, J., Brakke, K. A. & Berthier, E. Open Microfluidics (John Wiley & Sons, 2016).

  25. 25.

    Casavant, B. P. et al. Suspended microfluidics. Proc. Natl Acad. Sci. USA 110, 10111–10116 (2013).

    CAS  Article  Google Scholar 

  26. 26.

    Bishop, E. T. et al. An in vitro model of angiogenesis: basic features. Angiogenesis 3, 335–344 (1999).

    CAS  Article  Google Scholar 

  27. 27.

    Theberge, A. B. et al. Microfluidic multiculture assay to analyze biomolecular signaling in angiogenesis. Anal. Chem. 87, 3239–3246 (2015).

    CAS  Article  Google Scholar 

  28. 28.

    Sarkanen, J.-R. et al. Intra-laboratory pre-validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators. Front. Pharmacol. 1, 147 (2010).

    CAS  PubMed  Google Scholar 

  29. 29.

    Zhao, B., Moore, J. S. & Beebe, D. J. Surface-directed liquid flow inside microchannels. Science 291, 1023–1026 (2001).

    CAS  Article  Google Scholar 

  30. 30.

    Atencia, J. & Beebe, D. J. Controlled microfluidic interfaces. Nature 437, 648–655 (2005).

    CAS  Article  Google Scholar 

  31. 31.

    de Groot, T. E., Veserat, K. S., Berthier, E., Beebe, D. J. & Theberge, A. B. Surface-tension driven open microfluidic platform for hanging droplet culture. Lab Chip 16, 334–344 (2016).

    Article  Google Scholar 

  32. 32.

    Biederbick, A., Kern, H. F. & Elsässer, H. P. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur. J. Cell Biol. 66, 3–14 (1995).

    CAS  PubMed  Google Scholar 

  33. 33.

    Mosser, D. M. & Edwards, J. P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969 (2008).

    CAS  Article  Google Scholar 

  34. 34.

    Gordon, S. & Taylor, P. R. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953–964 (2005).

    CAS  Article  Google Scholar 

  35. 35.

    Mantovani, A. et al. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 25, 677–686 (2004).

    CAS  Article  Google Scholar 

  36. 36.

    Duluc, D. et al. Tumor-associated leukemia inhibitory factor and IL-6 skew monocyte differentiation into tumor-associated macrophage-like cells. Blood 110, 4319–4330 (2007).

    CAS  Article  Google Scholar 

  37. 37.

    Wu, H. C. et al. Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells. Int. J. Cancer 57, 406–412 (1994).

    CAS  Article  Google Scholar 

  38. 38.

    Kim, S., Lee, H., Chung, M. & Jeon, N. L. Engineering of functional, perfusable 3D microvascular networks on a chip. Lab Chip 13, 1489–1500 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Bersini, S. & Moretti, M. 3D functional and perfusable microvascular networks for organotypic microfluidic models. J. Mater. Sci. Mater. Med. 26, 180 (2015).

    Article  Google Scholar 

  40. 40.

    Guckenberger, D. J., de Groot, T. E., Wan, A. M. D., Beebe, D. J. & Young, E. W. K. Micromilling: a method for ultra-rapid prototyping of plastic microfluidic devices. Lab Chip 15, 2364–2378 (2015).

    CAS  Article  Google Scholar 

  41. 41.

    Chanput, W., Mes, J. J., Savelkoul, H. F. J. & Wichers, H. J. Characterization of polarized THP-1 macrophages and polarizing ability of LPS and food compounds. Food Funct. 4, 266–276 (2013).

    CAS  Article  Google Scholar 

  42. 42.

    Genin, M., Clement, F., Fattaccioli, A., Raes, M. & Michiels, C. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer 15, 577 (2015).

    Article  Google Scholar 

  43. 43.

    Tjiu, J.-W. et al. Tumor-associated macrophage-induced invasion and angiogenesis of human basal cell carcinoma cells by cyclooxygenase-2 induction. J. Invest. Dermatol. 129, 1016–1025 (2009).

    CAS  Article  Google Scholar 

  44. 44.

    Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work is funded by a National Science Foundation grant (EFRI-MKIS), University of Wisconsin Carbone Cancer Center Cancer Center Support Grant (P30 CA014520), Research Training in Hematology T32 (NIH T32 HL07899), NIH R01EB010039 BRG, NIH R01 CA185251, NIH K12 DK100022, DOD Prostate Cancer Research Program (W81XWH-16-0543), the Arnold and Mabel Beckman Foundation (Beckman Young Investigator Award), and the University of Washington. We thank D. Kosoff for helpful discussions and assistance with PCR, and undergraduate student B. Horman for facilitating experiments.

Author information

Affiliations

Authors

Contributions

J.Y., E.B., S.S., D.J.B. and A.B.T. designed the research. J.Y., A.C. and S.S. conducted experiments; all of the authors interpreted the data. J.Y., A.B.T., E.B. and D.J.B. wrote the manuscript, and all authors revised it.

Corresponding authors

Correspondence to David J. Beebe or Ashleigh B. Theberge.

Ethics declarations

Competing interests

The authors have potential conflicts of interest related to technologies presented here: J.Y. (Stacks to the Future), E.B. (Tasso, Salus Discovery and Stacks to the Future), T.E.d.G. (Stacks to the Future and Lynx Biosciences), A.B.T. (Stacks to the Future), and D.J.B. (Bellbrook Labs, Tasso, Stacks to the Future, Lynx Biosciences, Onexio Biosystems and Salus Discovery). However, none of these companies supported this work.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, methods and captions for Supplementary Videos 1–3.

Reporting summary

Supplementary Video 1

Stacks layers are open microfluidic devices that can be operated with a pipette.

Supplementary Video 2

Stacks devices can be assembled to enable diffusion.

Supplementary Video 3

Stacks devices are reconfigurable and enable diffusion across layers.

ImageJ macros

ImageJ macros for image processing.

Injection-moulding CAD designs

SolidWorks files for the Stack devices.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yu, J., Berthier, E., Craig, A. et al. Reconfigurable open microfluidics for studying the spatiotemporal dynamics of paracrine signalling. Nat Biomed Eng 3, 830–841 (2019). https://doi.org/10.1038/s41551-019-0421-4

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing