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.

Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro

Abstract

Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: The human small airway-on-a-chip.
Figure 2: Modeling asthma and lung inflammation on-chip.
Figure 3: Modeling COPD exacerbations in the small airway-on-a-chip.
Figure 4: Pharmacological modulation of IL-13 induced asthmatic phenotype and COPD exacerbation–associated inflammation in the small airway chip.
Figure 5: Therapeutic modulation of inflammatory cytokine and chemokine production in the COPD small airway chip.

References

  1. World Health Organization. Top 10 causes of death http://www.who.int/mediacentre/factsheets/fs310/en/ (2014).

  2. Bafadhel, M. . et al. Acute exacerbations of chronic obstructive pulmonary disease: identification of biologic clusters and their biomarkers. Am. J. Respir. Crit. Care Med. 184, 662–671 (2011).

    Article  PubMed  Google Scholar 

  3. Jackson, D.J., Sykes, A., Mallia, P. & Johnston, S.L. Asthma exacerbations: origin, effect, and prevention. J. Allergy Clin. Immunol. 128, 1165–1174 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Hyde, D.M., Hamid, Q. & Irvin, C.G. Anatomy, pathology, and physiology of the tracheobronchial tree: emphasis on the distal airways. J. Allergy Clin. Immunol. 124 (suppl. 6), S72–S77 (2009).

    Article  PubMed  Google Scholar 

  5. Kolaczkowska, E. & Kubes, P. Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 13, 159–175 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Wright, J.L., Cosio, M. & Churg, A. Animal models of chronic obstructive pulmonary disease. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L1–L15 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Di Stefano, A. et al. Severity of airflow limitation is associated with severity of airway inflammation in smokers. Am. J. Respir. Crit. Care Med. 158, 1277–1285 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Fahy, J.V. Eosinophilic and neutrophilic inflammation in asthma: insights from clinical studies. Proc. Am. Thorac. Soc. 6, 256–259 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Hogg, J.C. et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N. Engl. J. Med. 350, 2645–2653 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Bueters, T., Ploeger, B.A. & Visser, S.A. The virtue of translational PKPD modeling in drug discovery: selecting the right clinical candidate while sparing animal lives. Drug Discov. Today 18, 853–862 (2013).

    Article  PubMed  Google Scholar 

  11. Fulcher, M.L., Gabriel, S., Burns, K.A., Yankaskas, J.R. & Randell, S.H. Well-differentiated human airway epithelial cell cultures. Methods Mol. Med. 107, 183–206 (2005).

    CAS  PubMed  Google Scholar 

  12. Ugonna, K., Bingle, C.D., Plant, K., Wilson, K. & Everard, M.L. Macrophages are required for dendritic cell uptake of respiratory syncytial virus from an infected epithelium. PLoS ONE 9, e91855 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Didierlaurent, A. et al. Sustained desensitization to bacterial Toll-like receptor ligands after resolution of respiratory influenza infection. J. Exp. Med. 205, 323–329 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hammad, H. et al. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells. Nat. Med. 15, 410–416 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Teijaro, J.R. et al. Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection. Cell 146, 980–991 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Langer, H.F. & Chavakis, T. Leukocyte-endothelial interactions in inflammation. J. Cell. Mol. Med. 13, 1211–1220 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Esch, E.W., Bahinski, A. & Huh, D. Organs-on-chips at the frontiers of drug discovery. Nat. Rev. Drug Discov. 14, 248–260 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huh, D. et al. A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Sci. Transl. Med. 4, 159ra147 (2012).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  20. Huh, D. et al. Reconstituting organ-level lung functions on a chip. Science 328, 1662–1668 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wanner, A., Salathe, M. & O'Riordan, T.G. Mucociliary clearance in the airways. Am. J. Respir. Crit. Care Med. 154, 1868–1902 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Fliegauf, M., Benzing, T. & Omran, H. When cilia go bad: cilia defects and ciliopathies. Nat. Rev. Mol. Cell Biol. 8, 880–893 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Livraghi, A. & Randell, S.H. Cystic fibrosis and other respiratory diseases of impaired mucus clearance. Toxicol. Pathol. 35, 116–129 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Wills-Karp, M. Interleukin-13 in asthma pathogenesis. Immunol. Rev. 202, 175–190 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Kuperman, D.A. et al. Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat. Med. 8, 885–889 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Danahay, H. et al. Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung. Cell Rep. 10, 239–252 (2015).

    Article  CAS  PubMed  Google Scholar 

  27. Sousa, A.R., Poston, R.N., Lane, S.J., Nakhosteen, J.A. & Lee, T.H. Detection of GM-CSF in asthmatic bronchial epithelium and decrease by inhaled corticosteroids. Am. Rev. Respir. Dis. 147, 1557–1561 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. Ordoñez, C.L. et al. Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am. J. Respir. Crit. Care Med. 163, 517–523 (2001).

    Article  PubMed  Google Scholar 

  29. Thomas, B. et al. Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma. J. Allergy Clin. Immunol. 126, 722–729 (2010).

    Article  PubMed  Google Scholar 

  30. Hewson, C.A., Jardine, A., Edwards, M.R., Laza-Stanca, V. & Johnston, S.L. Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells. J. Virol. 79, 12273–12279 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Curradi, G. et al. Airway basal cell vascular endothelial growth factor-mediated cross-talk regulates endothelial cell-dependent growth support of human airway basal cells. Cell. Mol. Life Sci. 69, 2217–2231 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ding, B.S., Gomi, K., Rafii, S., Crystal, R.G. & Walters, M.S. Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells. J. Cell Sci. 128, 2983–2988 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Lawrence, M.B. & Springer, T.A. Neutrophils roll on E-selectin. J. Immunol. 151, 6338–6346 (1993).

    CAS  PubMed  Google Scholar 

  34. Mayer, A.K. et al. Differential recognition of TLR-dependent microbial ligands in human bronchial epithelial cells. J. Immunol. 178, 3134–3142 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. O'Shaughnessy, T.C., Ansari, T.W., Barnes, N.C. & Jeffery, P.K. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am. J. Respir. Crit. Care Med. 155, 852–857 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Papi, A. et al. Infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations. Am. J. Respir. Crit. Care Med. 173, 1114–1121 (2006).

    Article  PubMed  Google Scholar 

  37. Deslee, G. et al. Bronchial epithelial spheroids: an alternative culture model to investigate epithelium inflammation-mediated COPD. Respir. Res. 8, 86 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Fleischmann, R. et al. Placebo-controlled trial of tofacitinib monotherapy in rheumatoid arthritis. N. Engl. J. Med. 367, 495–507 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Bush, A. et al. Severe childhood asthma: a common international approach? Lancet 372, 1019–1021 (2008).

    Article  PubMed  Google Scholar 

  40. Kudlacz, E., Conklyn, M., Andresen, C., Whitney-Pickett, C. & Changelian, P. The JAK-3 inhibitor CP-690550 is a potent anti-inflammatory agent in a murine model of pulmonary eosinophilia. Eur. J. Pharmacol. 582, 154–161 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Ammit, A.J. Glucocorticoid insensitivity as a source of drug targets for respiratory disease. Curr. Opin. Pharmacol. 13, 370–376 (2013).

    Article  CAS  PubMed  Google Scholar 

  42. Wu, J. et al. Design and chemoproteomic functional characterization of a chemical probe targeted to bromodomains of BET family proteins. Medchemcomm 5, 1871–1878 (2014).

    Article  CAS  Google Scholar 

  43. Belkina, A.C., Nikolajczyk, B.S. & Denis, G.V. BET protein function is required for inflammation: Brd2 genetic disruption and BET inhibitor JQ1 impair mouse macrophage inflammatory responses. J. Immunol. 190, 3670–3678 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Abbassi, O., Kishimoto, T.K., McIntire, L.V., Anderson, D.C. & Smith, C.W. E-selectin supports neutrophil rolling in vitro under conditions of flow. J. Clin. Invest. 92, 2719–2730 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gomez-Cambronero, J., Horn, J., Paul, C.C. & Baumann, M.A. Granulocyte-macrophage colony-stimulating factor is a chemoattractant cytokine for human neutrophils: involvement of the ribosomal p70 S6 kinase signaling pathway. J. Immunol. 171, 6846–6855 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Hubeau, C., Kubera, J.E., Masek-Hammerman, K. & Williams, C.M. Interleukin-6 neutralization alleviates pulmonary inflammation in mice exposed to cigarette smoke and poly(I:C). Clin. Sci. (Lond.) 125, 483–493 (2013).

    Article  CAS  Google Scholar 

  47. Sellgren, K.L., Butala, E.J., Gilmour, B.P., Randell, S.H. & Grego, S. A biomimetic multicellular model of the airways using primary human cells. Lab Chip 14, 3349–3358 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Boers, J.E., Ambergen, A.W. & Thunnissen, F.B. Number and proliferation of clara cells in normal human airway epithelium. Am. J. Respir. Crit. Care Med. 159, 1585–1591 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Mercer, R.R., Russell, M.L., Roggli, V.L. & Crapo, J.D. Cell number and distribution in human and rat airways. Am. J. Respir. Cell Mol. Biol. 10, 613–624 (1994).

    Article  CAS  PubMed  Google Scholar 

  50. Villenave, R. et al. In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo. Proc. Natl. Acad. Sci. USA 109, 5040–5045 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Benam, K.H., Kok, W.L., McMichael, A.J. & Ho, L.P. Alternative spliced CD1d transcripts in human bronchial epithelial cells. PLoS ONE 6, e22726 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kitagawa, S., Takaku, F. & Sakamoto, S. Evidence that proteases are involved in superoxide production by human polymorphonuclear leukocytes and monocytes. J. Clin. Invest. 65, 74–81 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Funding was provided by Pfizer, Merck, Wyss Institute for Biologically Inspired Engineering at Harvard University and the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement Number W911NF-12-2-0036. We thank K. Karalis for helpful discussions, and B. Hassell and M. Mazur for technical assistance.

Author information

Authors and Affiliations

Authors

Contributions

K.H.B., R.V., C.H., H.-H.L., S.E.A., M.S., G.A.H. and D.E.I. designed the research; K.H.B. and R.V. developed the basic small airway chip model; R.V. and A.V. conducted the asthma work; K.H.B. and C.L. conducted the COPD studies; K.H.B. optimized and performed leukocyte-recruitment studies; J.C.W. performed scanning electron microscopy imaging; T.C.F. helped with confocal microscopy imaging; K.H.B. and R.V. prepared the manuscript; G.A.H. and A.B. commented on the manuscript; and D.E.I. critically revised the manuscript.

Corresponding author

Correspondence to Donald E Ingber.

Ethics declarations

Competing interests

D.E.I. and G.A.H. are founders and hold equity in Emulate, Inc., and D.E.I. chairs its scientific advisory board.

Integrated supplementary information

Supplementary Figure 1 Reconstitution of a differentiated human bronchiolar epithelium and pulmonary barrier on-chip.

(a) Well-differentiated human airway epithelium formed on-chip using hAECs derived from COPD donors; ciliated cells were labeled for β-tubulin IV (green), and goblet cells were stained for MUC5AC (magenta; scale bar, 20 μm; representative image from three independent stainings). (b) A confocal immunofluorescence image of club cells in bronchiolar epithelial cells differentiated on-chip (green, club cell secretory protein 10; yellow, F-actin; scale bar, 20 µm; representative image from two independent stainings). (c) Epithelial barrier function was assessed by flowing inulin-FITC (~4 kDa), dextran–Cascade blue (10 kDa) or dextran–Texas red (70 kDa) (100 µg ml−1 – 60 µL h−1) for 24h through the epithelial side of the small airway-on-a-chip containing endothelial cells alone or cocultured with well-differentiated hAECs or no cells, and measuring fluorescence in the effluent from the top and bottom channels. Barrier permeability is presented as apparent permeability (Papp; data from 1–2 independent biological replicates from 2 different donors are presented). (d) Transmission electron micrographic views of cilia formed on the apical surface of human airway epithelial cells grown in the small airway-on-a-chip; white arrows indicate two cilia (scale bar, 500 nm); inset shows a cross-section of an axoneme at higher magnification, highlighting the typical 9+2 structure (scale bar, 100 nm; representative image of 4 independent experiments performed using 4 different donors).

Source data

Supplementary Figure 2 Analysis of effects of the viral mimic poly(I:C) on interactions between epithelium and endothelium in the human small airway chip.

(a) GRO-α and IL-8 levels measured in basal secretions collected within the vascular effluent 24 h after fully differentiated hAECs cultured with (+) or without (–) endothelial cells in the presence (+) or absence (–) of 10 µg ml−1 poly(I:C) (unpaired Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001; data represent mean ± s.e.m (compared to unstimulated epithelium only) from 3 healthy donors, with 1 biological replicate per donor; n = 3). (b) Quantitative RT-PCR analysis of the effect of poly(I:C) (10 μg ml−1) stimulation of the small airway chip for 6 h on expression of endothelial genes encoding the cell-adhesion molecules VCAM-1 and E-selectin (unpaired Student’s t-test; data represent mean ± s.e.m. (compared to unstimulated) from one donor, with 3 biological replicates per condition; n = 3). (c) Comparison of poly(I:C) induced cytokine secretion from bronchiolar and bronchial epithelial cells on-chip. Note that comparable results were found for two key proinflammatory cytokines (unpaired Student’s t-test; *P < 0.05, **P < 0.01, n.s., not significant; data represent mean ± s.e.m (compared to unstimulated) from 3–5 different healthy donors, with 1–4 biological replicates per donor; n = 4–7).

Source data

Supplementary Figure 3 Modulation of cytokine and chemokine gene expression in COPD small airway chips using a BRD4 inhibitor.

Quantitative real-time PCR analysis of the effects of budesonide and BRD4 inhibitor (applied as described in Fig. 5) on expression of IL-8, MCP-1, IL-6 and GRO-α genes in lung blood microvascular endothelial cells lysed in situ within the microvascular channel of the chips (unpaired Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001; data represent mean ± s.e.m (compared to untreated) from two of the donors studied in Fig. 5, with 1–2 biological replicates per condition; n = 3).

Source data

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1 and 2, and Supplementary Discussion (PDF 855 kb)

3D visualization of the lung small airway epithelium and endothelium reconstituted on-chip.

A movie showing various confocal fluorescence microscopic 3D views of a fully differentiated, pseudostratified, human small airway epithelium cultured at an air-liquid interface, formed from primary hAECs (F-actin, green) and cocultured with primary human pulmonary microvascular endothelial cells (F-actin, red) in the top and lower channels of the small airway chip device, respectively (DAPI-stained nuclei, blue). (MOV 339 kb)

Z-stack reconstructed 3D visualization of the mucociliary small airway epithelium on-chip.

A video showing multiple confocal immunofluorescence 3D views of a polarized, mucociliary, human bronchiolar epithelium grown in the small airway chip showing cilia stained with anti−β-tubulin (cyan) and goblet cells labeled with anti-MUC5AC (magenta). (MOV 612 kb)

Active ciliary beating of the differentiated human airway epithelium on-chip.

Time-lapse video of phase-contrast views of the human small airway epithelium with apical cilia beating actively on-chip. The video is slowed down to enable analysis of cilia beating frequencies in the region of interest (square at top left). (MOV 757 kb)

Visualization of human airway epithelial mucociliary transport on-chip.

Real-time fluorescence microscopic imaging of mucociliary transport by the differentiated human airway epithelium cultured on-chip when exposed to fluorescent 1-μm-diameter microbeads (white; scale bar, 50 μm). (MOV 503 kb)

Recruitment and adhesion of circulating human leukocytes in the human small airway chip.

Real-time fluorescence microscopic imaging showing how freshly isolated, CellTracker red−labeled human neutrophils adhere to the endothelium and roll over its surface (a single adherent neutrophil is shown in this high-magnification view) when these leukocytes are flowed under physiological conditions (shear stress, 1 dyn cm−2) through the microvascular (endothelium-lined) channel of a human small airway chip that was stimulated by the addition of poly(I:C) to the epithelium in its upper channel. (AVI 1388 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Benam, K., Villenave, R., Lucchesi, C. et al. Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro. Nat Methods 13, 151–157 (2016). https://doi.org/10.1038/nmeth.3697

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.3697

This article is cited by

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