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

Journal name:
Nature Methods
Volume:
13,
Pages:
151–157
Year published:
DOI:
doi:10.1038/nmeth.3697
Received
Accepted
Published online

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.

At a glance

Figures

  1. The human small airway-on-a-chip.
    Figure 1: The human small airway-on-a-chip.

    (a) Schematic diagram of a cross-section through the small airway-on-a-chip. The dashed circle highlights the area depicted in b. (b) Diagram of the tissue-tissue interface that forms on-chip, showing differentiated airway epithelium (pink cells) cultured on top of a porous collagen-coated membrane at an air-liquid interface in the upper channel and the endothelium below (orange cells) with flowing medium that feeds both tissue layers. (c) A 3D reconstruction showing fully differentiated, pseudostratified airway epithelium formed on-chip by cultured hAECs (green, F-actin) with human pulmonary microvascular endothelial cells (red, F-actin) on the opposite side of the membrane. Blue denotes DAPI-stained nuclei; scale bar, 30 μm. (d) The differentiated human small airway epithelium exhibited continuous tight junctional connections on-chip, as demonstrated by ZO1 staining (red). Scale bar, 20 μm. (e) The human (lung blood microvascular) endothelial monolayer formed on-chip also contained continuous adherens junctions between adjacent cells, as indicated by PECAM-1 staining (green). Scale bar, 20 μm. (f) Well-differentiated human airway epithelium formed on-chip using hAECs derived from healthy donors demonstrating the presence of high numbers of ciliated cells labeled for β-tubulin IV (green) and goblet cells stained with anti-MUC5AC (magenta). Scale bar, 20 μm. (g) Scanning electron micrograph of cilia (blue) on the apical surface of the differentiated airway epithelium formed on-chip (nonciliated cells are in brown). Scale bar, 10 μm. (h) Sequential frames of a video of the apical surface of differentiated epithelium recorded over 100 ms showing cilia beating at a frequency of ~10 Hz. A single cilium is highlighted in white in each frame; time stamps and the white arrow indicate the duration and direction of one forward and return stroke (Supplementary Video 4 shows the full recording). Scale bar, 5 μm (applies to all images in panel). All images are representative of three to six independent experiments performed on cells from three to six different donors.

  2. Modeling asthma and lung inflammation on-chip.
    Figure 2: Modeling asthma and lung inflammation on-chip.

    (a) Immunofluorescence micrographic views of differentiated airway epithelium cultured on-chip for 4–6 weeks at the air-liquid interface in the absence (left) or presence (right) of IL-13 showing epithelium stained for the goblet cell marker MUC5AC (green) and with DAPI (blue). Scale bar, 50 μm (applies to both views); images are representative of three independent experiments performed on cells from three different donors. (bd) Total culture area covered by goblet cells (b), production of the cytokines G-CSF and GM-CSF (c) and cilia beating frequency (d) under the conditions described in a. Data represent mean and s.e.m. (compared to unstimulated controls) of cells from three healthy donors, with one or two biological replicates per donor; for goblet cell analysis, three to five representative areas per condition were used for quantification; n = 3–8. (e) Production of the cytokines RANTES, IL-6 and IP-10 by the small airway-on-a-chip with (+) or without (−) a differentiated bronchiolar epithelium (Epi), an umbilical vein endothelium (Endo) or both in the presence or absence of poly(I:C) (10 μg ml−1) stimulation; data represent mean and s.e.m. (compared to unstimulated epithelium or stimulated coculture) of cells from three or four healthy donors, with one biological replicate per donor; n = 3–4. Significance determined by unpaired Student's t-test; *P < 0.05, **P < 0.01, ***P < 0.001.

  3. Modeling COPD exacerbations in the small airway-on-a-chip.
    Figure 3: Modeling COPD exacerbations in the small airway-on-a-chip.

    The graphs show effects on production of the cytokines IL-8, M-CSF, IP-10 and RANTES by Toll-like receptor stimulation with LPS (10 μg ml−1) or poly(I:C) (10 μg ml−1) in small airway chips lined by 3–5-week-old hAECs obtained from either healthy or COPD subjects (all the COPD subjects had a history of smoking). Note that Toll-like receptor activation by these simulants of bacterial and viral infection significantly induced IL-8 and M-CSF release only in COPD chips, whereas IP-10 and RANTES release increased in both healthy and COPD epithelia (unpaired Student's t-test; *P < 0.05, **P < 0.01). Data represent mean and s.e.m. (compared to unstimulated controls) of cells from four different healthy and four different COPD donors, with one to three biological replicates (chips) per donor; n = 5–10. The variability between biological replicates for a given donor was minimal (s.e.m. <5% of mean).

  4. Pharmacological modulation of IL-13 induced asthmatic phenotype and COPD exacerbation-associated inflammation in the small airway chip.
    Figure 4: Pharmacological modulation of IL-13 induced asthmatic phenotype and COPD exacerbation–associated inflammation in the small airway chip.

    (a) Total area covered by goblet cells, effects on production of the cytokines G-CSF and GM-CSF, and cilia beating frequency after exposure of the epithelium to IL-13 with or without dexamethasone (Dex) or tofacitinib for 8 d (skewed cytokine data were log transformed before comparison; data represent mean and s.e.m. (compared to IL-13–treated condition) of cells from three healthy donors, with one or two biological replicates per donor; for goblet cell analysis, three to five representative areas per condition were used for quantification; n = 3–9). (b) Neutrophil-adhesion results compared with neutrophil-recruitment data obtained when similar studies were carried out in static Transwell cultures. Data represent mean and s.e.m. (chip versus Transwell) of cells from two to four different COPD donors, with one to four biological replicates per donor and three to ten independent fields of view per chip or Transwell; n = 15–59. Bud, budesonide; BRD4, bromodomain-containing protein 4. (c) Effects of (10 nM) budesonide and (500 nM) BRD4 inhibitor on expression of genes encoding the endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1, compared to the untreated (0.1% DMSO) group, as measured using real-time PCR; human pulmonary blood microvascular cells were studied (n.s., not significant; data represent mean and s.e.m. of cells from one of the donors studied in b; three or four biological replicates per condition; n = 3–4). Significance determined by unpaired Student's t-test; *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant.

  5. Therapeutic modulation of inflammatory cytokine and chemokine production in the COPD small airway chip.
    Figure 5: Therapeutic modulation of inflammatory cytokine and chemokine production in the COPD small airway chip.

    Analysis of the effects of budesonide (Bud) and BRD4 inhibitor on the secretion of IL-8, MCP-1, GRO-α, IL-6 and GM-CSF into the microvascular channel of chips lined by COPD epithelium cultured at the air-liquid interface for 3–5 weeks when challenged with poly(I:C) (unpaired Student's t-test; *P < 0.05, **P < 0.01, ***P < 0.001; data represent mean and s.e.m. (compared to untreated controls) of cells from three different COPD epithelial and two different lung blood microvascular endothelial cell donors, with one to four biological replicates per donor; n = 6–8).

  6. Reconstitution of a differentiated human bronchiolar epithelium and pulmonary barrier on-chip.
    Supplementary Fig. 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).

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

  8. Modulation of cytokine and chemokine gene expression in COPD small airway chips using a BRD4 inhibitor.
    Supplementary Fig. 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).

Videos

  1. 3D visualization of the lung small airway epithelium and endothelium reconstituted on-chip.
    Video 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. Z-stack reconstructed 3D visualization of the mucociliary small airway epithelium on-chip.
    Video 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. Active ciliary beating of the differentiated human airway epithelium on-chip.
    Video 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. Visualization of human airway epithelial mucociliary transport on-chip.
    Video 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. Recruitment and adhesion of circulating human leukocytes in the human small airway chip.
    Video 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.

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, 662671 (2011).
  3. Jackson, D.J., Sykes, A., Mallia, P. & Johnston, S.L. Asthma exacerbations: origin, effect, and prevention. J. Allergy Clin. Immunol. 128, 11651174 (2011).
  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), S72S77 (2009).
  5. Kolaczkowska, E. & Kubes, P. Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 13, 159175 (2013).
  6. Wright, J.L., Cosio, M. & Churg, A. Animal models of chronic obstructive pulmonary disease. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L1L15 (2008).
  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, 12771285 (1998).
  8. Fahy, J.V. Eosinophilic and neutrophilic inflammation in asthma: insights from clinical studies. Proc. Am. Thorac. Soc. 6, 256259 (2009).
  9. Hogg, J.C. et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N. Engl. J. Med. 350, 26452653 (2004).
  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, 853862 (2013).
  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, 183206 (2005).
  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).
  13. Didierlaurent, A. et al. Sustained desensitization to bacterial Toll-like receptor ligands after resolution of respiratory influenza infection. J. Exp. Med. 205, 323329 (2008).
  14. Hammad, H. et al. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells. Nat. Med. 15, 410416 (2009).
  15. Teijaro, J.R. et al. Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection. Cell 146, 980991 (2011).
  16. Langer, H.F. & Chavakis, T. Leukocyte-endothelial interactions in inflammation. J. Cell. Mol. Med. 13, 12111220 (2009).
  17. Bhatia, S.N. & Ingber, D.E. Microfluidic organs-on-chips. Nat. Biotechnol. 32, 760772 (2014).
  18. Esch, E.W., Bahinski, A. & Huh, D. Organs-on-chips at the frontiers of drug discovery. Nat. Rev. Drug Discov. 14, 248260 (2015).
  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).
  20. Huh, D. et al. Reconstituting organ-level lung functions on a chip. Science 328, 16621668 (2010).
  21. Wanner, A., Salathe, M. & O'Riordan, T.G. Mucociliary clearance in the airways. Am. J. Respir. Crit. Care Med. 154, 18681902 (1996).
  22. Fliegauf, M., Benzing, T. & Omran, H. When cilia go bad: cilia defects and ciliopathies. Nat. Rev. Mol. Cell Biol. 8, 880893 (2007).
  23. Livraghi, A. & Randell, S.H. Cystic fibrosis and other respiratory diseases of impaired mucus clearance. Toxicol. Pathol. 35, 116129 (2007).
  24. Wills-Karp, M. Interleukin-13 in asthma pathogenesis. Immunol. Rev. 202, 175190 (2004).
  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, 885889 (2002).
  26. Danahay, H. et al. Notch2 is required for inflammatory cytokine-driven goblet cell metaplasia in the lung. Cell Rep. 10, 239252 (2015).
  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, 15571561 (1993).
  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, 517523 (2001).
  29. Thomas, B. et al. Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma. J. Allergy Clin. Immunol. 126, 722729 (2010).
  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, 1227312279 (2005).
  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, 22172231 (2012).
  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, 29832988 (2015).
  33. Lawrence, M.B. & Springer, T.A. Neutrophils roll on E-selectin. J. Immunol. 151, 63386346 (1993).
  34. Mayer, A.K. et al. Differential recognition of TLR-dependent microbial ligands in human bronchial epithelial cells. J. Immunol. 178, 31343142 (2007).
  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, 852857 (1997).
  36. Papi, A. et al. Infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations. Am. J. Respir. Crit. Care Med. 173, 11141121 (2006).
  37. Deslee, G. et al. Bronchial epithelial spheroids: an alternative culture model to investigate epithelium inflammation-mediated COPD. Respir. Res. 8, 86 (2007).
  38. Fleischmann, R. et al. Placebo-controlled trial of tofacitinib monotherapy in rheumatoid arthritis. N. Engl. J. Med. 367, 495507 (2012).
  39. Bush, A. et al. Severe childhood asthma: a common international approach? Lancet 372, 10191021 (2008).
  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, 154161 (2008).
  41. Ammit, A.J. Glucocorticoid insensitivity as a source of drug targets for respiratory disease. Curr. Opin. Pharmacol. 13, 370376 (2013).
  42. Wu, J. et al. Design and chemoproteomic functional characterization of a chemical probe targeted to bromodomains of BET family proteins. Medchemcomm 5, 18711878 (2014).
  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, 36703678 (2013).
  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, 27192730 (1993).
  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, 68466855 (2003).
  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, 483493 (2013).
  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, 33493358 (2014).
  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, 15851591 (1999).
  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, 613624 (1994).
  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, 50405045 (2012).
  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, 402408 (2001).
  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).
  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, 7481 (1980).

Download references

Author information

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

    • Carolina Lucchesi,
    • Antonio Varone &
    • Geraldine A Hamilton
  2. These authors contributed equally to this work.

    • Kambez H Benam &
    • Remi Villenave

Affiliations

  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

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.

Competing financial 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:

Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Reconstitution of a differentiated human bronchiolar epithelium and pulmonary barrier on-chip. (500 KB)

    (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).

  2. 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. (117 KB)

    (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).

  3. Supplementary Figure 3: Modulation of cytokine and chemokine gene expression in COPD small airway chips using a BRD4 inhibitor. (103 KB)

    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).

Video

  1. Video 1: 3D visualization of the lung small airway epithelium and endothelium reconstituted on-chip. (339 KB, Download)
    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. Video 2: Z-stack reconstructed 3D visualization of the mucociliary small airway epithelium on-chip. (613 KB, Download)
    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. Video 3: Active ciliary beating of the differentiated human airway epithelium on-chip. (757 KB, Download)
    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. Video 4: Visualization of human airway epithelial mucociliary transport on-chip. (504 KB, Download)
    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. Video 5: Recruitment and adhesion of circulating human leukocytes in the human small airway chip. (1.35 MB, Download)
    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.

PDF files

  1. Supplementary Text and Figures (876 KB)

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

Additional data