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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- 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).
- 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).
- 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: 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).
- 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).
- 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).
- 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).
- 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.
- Supplementary Text and Figures (876 KB)
Supplementary Figures 1–3, Supplementary Tables 1 and 2, and Supplementary Discussion