Fig. 2 On-chip formation and mechanical stretching of an alveolar-capillary interface. (A) Long-term microfluidic coculture produces a tissue-tissue interface consisting of a single layer of the alveolar epithelium (epithelium, stained with CellTracker Green) closely apposed to a monolayer of the microvascular endothelium (endothelium, stained with CellTracker Red), both of which express intercellular junctional structures stained with antibodies to occludin or VE-cadherin, separated by a flexible ECM-coated PDMS membrane. Scale bar, 50 μm. (B) Surfactant production by the alveolar epithelium during air-liquid interface culture in our device detected by cellular uptake of the fluorescent dye quinacrine that labels lamellar bodies (white dots). Scale bar, 25 μm. (C) Air-liquid interface (ALI) culture leads to a greater increase in transbilayer electrical resistance (TER) and produces tighter alveolar-capillary barriers with higher TER (>800 Ω·cm2), as compared with the tissue layers formed under submerged liquid culture conditions. (D) Alveolar barrier permeability measured by quantitating the rate of fluorescent albumin transport is significantly reduced in ALI cultures compared with liquid cultures (*P < 0.001). Data in (C) and (D) represent the mean ± SEM from three separate experiments. (E) Membrane stretching–induced mechanical strain visualized by the displacements of individual fluorescent quantum dots that were immobilized on the membrane in hexagonal and rectangular patterns before (red) and after (green) stretching. Scale bar, 100 μm. (F) Membrane stretching exerts tension on the cells and causes them to distort in the direction of the applied force, as illustrated by the overlaid outlines of a single cell before (blue) and after (red) application of 15% strain. The pentagons in the micrographs represent microfabricated pores in the membrane. Endothelial cells were used for visualization of cell stretching.

(A) Ultrafine silica nanoparticles introduced through an air-liquid interface overlying the alveolar epithelium induce ICAM-1 expression (red) in the underlying endothelium and adhesion of circulating neutrophils (white dots) in the lower channel. Scale bar, 50 μm. Graph shows that physiological mechanical strain and silica nanoparticles synergistically up-regulate ICAM-1 expression (*P < 0.005; **P < 0.001). (B) Alveolar epithelial cells increase ROS production when exposed to silica nanoparticles (100 μg/ml) in conjunction with 10% cyclic strain (square) (P < 0.0005), whereas nanoparticles (triangle) or strain (diamond) alone had no effect on intracellular ROS levels relative to control cells (circle); ROS generation was normalized to the mean ROS value at time 0. (C) The alveolar epithelium responds to silica nanoparticles in a strain-dependent manner (*P < 0.001). (D) Addition of 50-nm superparamagnetic nanoparticles produced only a transient elevation of ROS in the epithelial cells subjected to 10% cyclic strain (P < 0.0005). (E) Application of physiological mechanical strain (10%) promotes increased cellular uptake of 100-nm polystyrene nanoparticles (magenta) relative to static cells, as illustrated by representative sections (a to d) through fluorescent confocal images. Internalized nanoparticles are indicated with arrows; green and blue show cytoplasmic and nuclear staining, respectively. (F) Transport of nanomaterials across the alveolar-capillary interface of the lung is simulated by nanoparticle transport from the alveolar chamber to the vascular channel of the lung mimic device. (G) Application of 10% mechanical strain (closed square) significantly increased the rate of nanoparticle translocation across the alveolar-capillary interface compared with static controls in this device (closed triangle) or in a Transwell culture system (open triangle) (P < 0.0005). (H) Fluorescence micrographs of a histological section of the whole lung showing 20-nm fluorescent nanoparticles (white dots, indicated with arrows in the inset at upper right that shows the region enclosed by the dashed square at higher magnification) present in the lung after intratracheal injection of nebulized nanoparticles and ex vivo ventilation in the mouse lung model. Nanoparticles cross the alveolar-capillary interface and are found on the surface of the alveolar epithelium, in the interstitial space, and on the capillary endothelium. PC, pulmonary capillary; AS, alveolar space; blue, epithelial nucleus; scale bar, 20 μm. (I) Physiological cyclic breathing generated by mechanical ventilation in whole mouse lung produces an increase by a factor of more than 5-fold in nanoparticle absorption into the blood perfusate when compared to lungs without lung ventilation (P < 0.0005). The graph indicates the number of nanoparticles detected in the pulmonary blood perfusate over time, as measured by drying the blood (1 μl) on glass and quantitating the number of particles per unit area (0.5 mm2). (J) The rate of nanoparticle translocation was significantly reduced by adding NAC to scavenge free radicals (*P < 0.001).

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Fig. 3

Fig. 1 Biologically inspired design of a human breathing lung-on-a-chip microdevice. (A) The microfabricated lung mimic device uses compartmentalized PDMS microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with ECM. The device recreates physiological breathing movements by applying vacuum to the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar-capillary barrier. (B) During inhalation in the living lung, contraction of the diaphragm causes a reduction in intrapleural pressure (Pip), leading to distension of the alveoli and physical stretching of the alveolar-capillary interface. (C) Three PDMS layers are aligned and irreversibly bonded to form two sets of three parallel microchannels separated by a 10-μm-thick PDMS membrane containing an array of through-holes with an effective diameter of 10 μm. Scale bar, 200 μm. (D) After permanent bonding, PDMS etchant is flowed through the side channels. Selective etching of the membrane layers in these channels produces two large side chambers to which vacuum is applied to cause mechanical stretching. Scale bar, 200 μm. (E) Images of an actual lung-on-a-chip microfluidic device viewed from above.