Fig. 3.

Reconstitution and direct visualization of complex organ-level responses involved in pulmonary inflammation and infection in the lung-on-a-chip device. (A) Epithelial stimulation with TNF-α (50 ng/ml) upregulates ICAM-1 expression (red) on the endothelium; control shows lack of ICAM-1 expression in the absence of TNF-α treatment. Cells were stretched with 10% strain at 0.2 Hz in both cases. (B) Fluorescently-labeled human neutrophils (white dots) avidly to the activated endothelium within one minute after introduction into the vascular channel. (C) Time-lapse microscopic images showing a captured neutrophil (white arrow) that spreads via firm adhesion and then crawls over the apical surface of the activated endothelium (not visible in this view; direction indicated by yellow arrows) until it forces itself through the cell-cell boundary within about 2 minutes after adhesion (times indicated in seconds). During the following 3 to 4 minutes, the neutrophil transmigrates through the alveolar-capillary barrier by passing through a pentagonal pore in the PDMS membrane, and then it moves away from the focal plane, causing it to appear blurry in the micrographs. (D) Phase contrast microscopic images show a neutrophil (arrow) emerging from the apical surface of the alveolar epithelium at the end of its transmigration over a period of approximately 3 minutes; thus, complete passage takes approximately 6 minutes in total. (E) Time-lapse fluorescence microscopic images showing phagocytosis of two GFP-expressing E. coli (green) bacteria on the epithelial surface by a neutrophil (red) that transmigrated from the vascular microchannel to the alveolar compartment. (bar, 50 μm in (A), (B) and 20 μm in (C)-(E)).