A Model of Human Small Airway on a Chip for Studies of Subacute Effects of Inhalation Toxicants

Abstract

Testing for acute inhalation hazards is conducted in animals; however, a number of robust in vitro human cell-based alternatives have been developed and tested. These models range in complexity from cultures of cell lines or primary cells in air-liquid interface on Transwells, to more complex and physiologically relevant flow- and mechanical stimulation-enabled tissue chips. Although the former models are relatively straightforward to establish and can be tested in medium/high throughput, the latter require specialized equipment and lack in throughput. In this study, we developed a device that can be easily manufactured while allowing for the production of a differentiated lung tissue. This multilayered microfluidic device enables coculture of primary human small airway epithelial cells and lung microvascular endothelial cells under physiological conditions for up to 18 days and recreates the parenchymal-vascular interface in the distal lung. To explore the potential of this airway on a chip for applications in inhalation toxicology, we also devised a system that allows for direct gas/aerosol exposures of the engineered airway epithelium to noxious stimuli known to cause adverse respiratory effects, including dry flowing air, lipopolysaccharide, particulate matter, and iodomethane. This study generated quantitative, high-content data that were indicative of aberrant changes in biochemical (lactate dehydrogenase), barrier (dextran permeability), functional (ciliary beating), and molecular (imaging for various markers) phenotypes of the small airway epithelium due to inhalational exposures. This study is significant because it established an in vitro model of human small airway on a chip that can be used in medium/high-throughput studies of subacute effects of inhalation toxicants.

Keywords: in vitro models; hazard identification; lung.

Figure 1.

Basic structure and function of the “closed” small airway epithelium tissue chip and response to the flowing dry air. A, Device configuration scaled to the U.S. quarter dollar coin. Visible are cell culture reservoirs for 2 chambers separated by a porous membrane. B, Schematic diagram of cross-section of the cell positioning in the device. See Supplementary Figure 1 for detailed images and chip design file. Top chamber contains human small airway epithelial cells (SAEC) and can be either filled with media or contain humidified air; bottom chamber contains human micro-vascular endothelial cells derived from lung (HMVEC-L) in cell culture media. Chamber dimensions are indicated. C, Typical timeline for model establishment and testing. Main milestones of device manipulation are shown. D, Representative images (4–10×) of the device with cells at day 15 of the experiment. Cells were stained (live for Calcein and fixed for other markers) and imaged. E, FITC-dextran (70 kDa) permeability was tested when the solution was added to the SAEC chamber. Fluorescence was quantified in both chambers after 15 min. The asterisk (*) indicates a significant difference (p < .05) between conditions using a t test. F, Daily measurements of LDH and mucin-1 were conducted in cell culture media from the HMVEC-L chamber. Shaded area and filled squares indicate days at which the incubator chamber had reduced humidity. G, Representative image of the fluorescent bead movement (10 s trace) in the SAEC chamber of a device cultured in ALI for 14 days.

Figure 2.

Effects of lipopolysaccharide (LPS) and PM2.5 on the small airway epithelium tissue chip. A, Effects of the LPS added to the HMVEC-L chamber (1 µg/ml was added to the HMVEC-L media daily for 7 days). The devices were established over 14 days and then LPS treatments (for 7 additional days) were conducted. Cell barrier was evaluated as FITC-dextran (70 kDa, 15 min) permeability; the red dotted line indicates cell permeability of the device without cells. SAEC function was evaluated by the ciliary movement (angle of divergence and bead velocity) without and LPS treatment. The asterisk (*) indicates a significant difference (p < .05) between conditions using a t test. B and C, Effects of the LPS (1 µg/ml for 5 h) or PM2.5 (10 µg/ml for 5 h) that were added to the SAEC chamber. ALI chambers were submerged with SAEC media without or with treatments for 5 h at which point it was removed without rinsing and the devices were incubated at ALI overnight. In some devices, cells were stained for Calcein (live cells), ZO-1 (tight junctions), and Hoechst (DNA); in other devices, fluorescent beads were added to the SAEC chamber and bead movement was tracked for 10 s. Representative images of ZO-1 and Hoechst staining, or bead traces are shown in (B). Quantification of Calcein and ZO-1 staining, and bead velocity are shown in (C).

Figure 3.

Basic structure and function of the “open” small airway epithelium tissue chip and exposure to dry flowing air. A, Device configuration scaled to the U.S. quarter dollar coin. Visible are cell culture reservoirs for 2 chambers separated by a porous membrane; PDMS was removed from the top layer to open the SAEC chamber to the surrounding environment. B, Representative images (10–40×) of the closed and open devices with cells at day 17 of the experiment. SAEC were stained (live for Calcein and fixed for other markers) and imaged. Molecular markers that were examined are shown. Both types of devices were kept in custom-made exposure chambers under constant 10 ml/min airflow. C, A representative cross-section of a z-stack (15 µm total, 40 slices) of the confocal images taken on the devices as detailed in (B). D, Cell height [measured from z stacks in (C)] in open versus closed chip designs. Box (interquartile range) and whisker (min-max range) plots show the individual measurements across cross-sections and devices. The asterisk (*) indicates a significant difference (p < .05) between conditions using a t test. E, Periodic measurements of LDH were conducted in cell culture media from the HMVEC-L chamber in open (squares) and closed (circles) tissue chips maintained under 10 ml/min air flow. F, Relative cell viability as a function of air flow and chip design. Closed design (black bars) with no airflow was used as a reference. Asterisks (*) denote a significant difference (p < .05) between conditions using a t test at each air flow condition.

Figure 4.

Performance of the “open” small airway epithelium tissue chip without airflow in a tissue culture incubator, or with airflow in a custom incubated exposure chamber. Devices were either kept in a standard tissue culture incubator (0 ml/min) or placed into a custom exposure chamber kept in a heated (37°C) chamber placed in a chemical fume hood (see Materials and Methods) under 5 ml/min flow of the humidified 5% CO₂-containing air for 5 h. A, Representative images of SAEC chamber stained for ZO-1 (red stain) and Hoechst (blue). B, Cell viability (from CellTiterGlo assay on SAECs), LDH activity (in cell culture media in the HMVEC-L channel), ZO-1 staining [quantified from the images shown in (A)], and permeability (from FITC-dextran, 70 kDa). Asterisks indicate a significant difference (*, p < .05; **, p < .01) between conditions using a t test (LDH assay) or one-way analysis of variance (for permeability).

Figure 5.

Effects of iodomethane exposure on the “open” small airway epithelium tissue chip. Devices were either kept in a custom exposure chamber kept in a heated (37°C) chamber placed in a chemical fume hood (see Materials and Methods) under 5 ml/min flow of the humidified 5% CO₂-containing air for 5 h without (0 ppm) or with (500 or 1000 ppm) iodomethane. A, Representative images of SAEC chamber stained for ZO-1 (red stain) and Hoechst (blue). B, Cell viability (from CellTiterGlo assay on SAECs), LDH activity (in cell culture media in the HMVEC-L channel), ZO-1 staining [quantified from the images shown in (A)], and permeability (from FITC-dextran, 70 kDa). Asterisks indicate a significant difference (*, p < .05; **, p < .01) among conditions using one-way analysis of variance.