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

Nature Methods volume 13, pages 151157 (2016) | Download Citation


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.

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  1. 1.

    World Health Organization. Top 10 causes of death (2014).

  2. 2.

    . 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).

  3. 3.

    , , & Asthma exacerbations: origin, effect, and prevention. J. Allergy Clin. Immunol. 128, 1165–1174 (2011).

  4. 4.

    , & Anatomy, pathology, and physiology of the tracheobronchial tree: emphasis on the distal airways. J. Allergy Clin. Immunol. 124 (suppl. 6), S72–S77 (2009).

  5. 5.

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

  6. 6.

    , & Animal models of chronic obstructive pulmonary disease. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L1–L15 (2008).

  7. 7.

    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).

  8. 8.

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

  9. 9.

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

  10. 10.

    , & 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).

  11. 11.

    , , , & Well-differentiated human airway epithelial cell cultures. Methods Mol. Med. 107, 183–206 (2005).

  12. 12.

    , , , & Macrophages are required for dendritic cell uptake of respiratory syncytial virus from an infected epithelium. PLoS ONE 9, e91855 (2014).

  13. 13.

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

  14. 14.

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

  15. 15.

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

  16. 16.

    & Leukocyte-endothelial interactions in inflammation. J. Cell. Mol. Med. 13, 1211–1220 (2009).

  17. 17.

    & Microfluidic organs-on-chips. Nat. Biotechnol. 32, 760–772 (2014).

  18. 18.

    , & Organs-on-chips at the frontiers of drug discovery. Nat. Rev. Drug Discov. 14, 248–260 (2015).

  19. 19.

    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).

  20. 20.

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

  21. 21.

    , & Mucociliary clearance in the airways. Am. J. Respir. Crit. Care Med. 154, 1868–1902 (1996).

  22. 22.

    , & When cilia go bad: cilia defects and ciliopathies. Nat. Rev. Mol. Cell Biol. 8, 880–893 (2007).

  23. 23.

    & Cystic fibrosis and other respiratory diseases of impaired mucus clearance. Toxicol. Pathol. 35, 116–129 (2007).

  24. 24.

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

  25. 25.

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

  26. 26.

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

  27. 27.

    , , , & Detection of GM-CSF in asthmatic bronchial epithelium and decrease by inhaled corticosteroids. Am. Rev. Respir. Dis. 147, 1557–1561 (1993).

  28. 28.

    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).

  29. 29.

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

  30. 30.

    , , , & 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).

  31. 31.

    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).

  32. 32.

    , , , & Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells. J. Cell Sci. 128, 2983–2988 (2015).

  33. 33.

    & Neutrophils roll on E-selectin. J. Immunol. 151, 6338–6346 (1993).

  34. 34.

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

  35. 35.

    , , & 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).

  36. 36.

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

  37. 37.

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

  38. 38.

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

  39. 39.

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

  40. 40.

    , , , & 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).

  41. 41.

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

  42. 42.

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

  43. 43.

    , & 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).

  44. 44.

    , , , & E-selectin supports neutrophil rolling in vitro under conditions of flow. J. Clin. Invest. 92, 2719–2730 (1993).

  45. 45.

    , , & 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).

  46. 46.

    , , & Interleukin-6 neutralization alleviates pulmonary inflammation in mice exposed to cigarette smoke and poly(I:C). Clin. Sci. (Lond.) 125, 483–493 (2013).

  47. 47.

    , , , & A biomimetic multicellular model of the airways using primary human cells. Lab Chip 14, 3349–3358 (2014).

  48. 48.

    , & Number and proliferation of clara cells in normal human airway epithelium. Am. J. Respir. Crit. Care Med. 159, 1585–1591 (1999).

  49. 49.

    , , & Cell number and distribution in human and rat airways. Am. J. Respir. Cell Mol. Biol. 10, 613–624 (1994).

  50. 50.

    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).

  51. 51.

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

  52. 52.

    , , & Alternative spliced CD1d transcripts in human bronchial epithelial cells. PLoS ONE 6, e22726 (2011).

  53. 53.

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

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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

Author notes

    • Carolina Lucchesi
    • , Antonio Varone
    •  & Geraldine A Hamilton

    Present address: Emulate Inc., Cambridge, Massachusetts, USA.

    • Kambez H Benam
    •  & Remi Villenave

    These authors contributed equally to this work.


  1. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.

    • Kambez H Benam
    • , Remi Villenave
    • , Carolina Lucchesi
    • , Antonio Varone
    • , Thomas C Ferrante
    • , James C Weaver
    • , Anthony Bahinski
    • , Geraldine A Hamilton
    •  & Donald E Ingber
  2. Pfizer, Cambridge, Massachusetts, USA.

    • Cedric Hubeau
  3. Merck Research Laboratories, Boston, Massachusetts, USA.

    • Hyun-Hee Lee
    • , Stephen E Alves
    •  & Michael Salmon
  4. Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.

    • James C Weaver
    •  & Donald E Ingber
  5. Vascular Biology Program, Boston Children's Hospital, Boston, Massachusetts, USA.

    • Donald E Ingber
  6. Harvard Medical School, Harvard University, Boston, Massachusetts, USA.

    • Donald E Ingber


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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.

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.

Corresponding author

Correspondence to Donald E Ingber.

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–3, Supplementary Tables 1 and 2, and Supplementary Discussion


  1. 1.

    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).

  2. 2.

    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).

  3. 3.

    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).

  4. 4.

    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).

  5. 5.

    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.

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