- Apr 2023
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tanyerilab.net tanyerilab.net
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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.
Questions: Does the lung-on-a-chip device function like a normal human alveolar interface? Do the cells grown on the PDMS membrane demonstrate the integrity, viability, and permeability of the typical cell layers found in the lungs?
Methods: Proper growth of cell layers on the PDMS membrane is verified with the fluorescent imaging of the red endothelial cells (that line the alveolar-capillary interface) and the green epithelial cells (that line the alveolar-air interface). Cell-cell tethering is a measure of membrane viability, and is demonstrated with imaging of Occludin and VE Cadherin, two proteins responsible for linking cells together. Typical alveolar cell junctions also bind relatively tightly and produce a surfactant that helps keep airways open during breathing, so electrical resistance readings and fluorescent imaging were used to confirm membrane viability. The human alveolar membrane is usually permeable to immune cells and other proteins, so the group measured the migration of fluorescently-labeled proteins across the PDMS barrier.
Conclusions: The lung-on-a-chip device demonstrated the typical behavior of human alveolar cell membranes when slower-than normal "breathing" was simulated.
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- Mar 2023
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tanyerilab.net tanyerilab.net
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(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).
A./B./C. Ultrafine silica particles are introduced to the lung-on-a-chip while a 10% cyclic strain is applied to the alveolar capillary interface. The authors observed ICAM-1 expression and neutrophil adhesion on the endothelial (capillary) surface, and measured the reactive oxygen species in the absence and presence of applied mechanical strain. The authors found out that applied mechanical strain increases inflammatory response (ICAM-1 expression, neutrophil adhesion, and ROS production) D. The same procedure described above was applied using superparamagnetic nanoparticles, another irritant that can be harmful to the human lung. This led to only a transient increase in ROS production, indicating a less toxic outcome. E./F./G The authors studied uptake of 100nm polystyrene nanoparticles by the lung mimic device compared to a 2D culture. The transwell system is the gold standard in current toxicology studies, where cells are cultured on two sides of a porous, static membrane. The results show that when mechanical strain is added, the lung-on-a-chip culture uptakes much more nanoparticles than the static transwell culture. H./I./J. In these experiments, the authors compare results to a mouse model. The authors observed that whole mouse lung subject to mechanical ventilation shows a 5-fold increase in nanoparticle absorption compared to lungs without ventilation. These results confirm that lung-on-a-chip device subject to mechanical strain is a better replica of the human lung compared to a 2D static culture.
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Fig. 4
In this experiment, the authors wanted to put the lung-on-a-chip to the ultimate test: simulating a real toxicology study! The group exposed the 'breathing' lung on chip device to silica nanoparticles. The group quantified the increase in inflammatory response (ICAM-1 expression and ROS production), and the uptake of nanoparticles across the alveolar-capillary interface. They then compared these results with those from a 2D static cell culture study and an animal study (mice). The group found out that the application of mechanical stress results in a drastically higher inflammatory response (increase in ICAM-1 and ROS) and uptake of nanoparticles in the lung-on-chip than the 2D static culture. The results demonstrate the effectiveness of their device in mimicking human lung, which might replace the mice models in the future.
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- Feb 2023
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tanyerilab.net tanyerilab.net
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Fig. 3
Questions: Can the lung-on-a-chip device produce an immune response like normal immune cells would in the lungs?
Methods: immune cells and proteins that trigger an immune cell response were exposed to the cells on the chip's membrane. The adhesion and migration of the immune cells on the alveolar membrane was shown with microscopic images. The immune response and inflammation of the alveolar cells was shown to occur within 6 minutes. Lastly, the lung-on-a-chip was exposed to a bacterial infection and immune response was measured like before.
results: The immune response of the lung-on-a-chip device mimic the response found in the human lung. When infected with bacteria, the alveolar layers deployed the appropriate immune cells in a timely fashion. This means that the lung-on-a-chip can effectively mimic the immune response of the human lung.
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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.
Question(s) - Can we create a chambered device that mimics the small pockets of tissues in the lungs where oxygen diffuses into the blood? Can we fabricate this device so that it reliably and accurately demonstrates the inhaling and exhaling of lungs? Will the device have similar integrity, viability, and permeability to typical alveolar-capillary interfaces found in humans?
Methods: A layer of PDMS is sandwiched between two halves of a molded chamber to make three parallel air pathways on the chip. the same type of epithelial and endothelial cells found in the linings of a human lung are cultured onto opposite sides of the PDMS membrane, and air is pumped in and out of the chambers in the same way that air goes into and out of the lungs during breathing.
results - the "inhaling" and "exhaling" air channels are colored blue and red on a fully-functional lung-on-a-chip device, and cells grown on the PDMS layer actively undergo the same mechanical stresses that their real-life counterparts experience in the human body. The entire lung-on-a-chip system measures roughly 1-2cm long, and external pumping mechanisms drive air in and out of the device.
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