Gravity-perfused airway-on-a-chip optimized for quantitative BSL-3 studies of SARS-CoV-2 infection: barrier permeability, cytokine production, immunohistochemistry, and viral load assays

Abstract

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.

Fig. 1. Layout and design of airway chip for BSL-3 use. A) A schematic representation of a gravity-perfused airway chip with two chambers separated by a PET membrane (dashed line) and bonded inside the PDMS (hatched), with both chambers perfused by the difference in height of the media in the supply and collection chambers. B) An airway chip with the epithelial cells on the upper surface of the barrier membrane providing an air–liquid interface. C) An illustration of the complete chip. D) An exploded drawing of the device. E) The mask layouts for the vascular (red) and airway (green) chambers and perfusion channels. F) A photograph of a completed airway chip with gravity reservoirs for use in a BSL-3 space.

Fig. 2. Fabrication of the gravity-perfused airway chip. A) An exploded drawing of the mold used to cast the deep supply and collection reservoirs. B) Details of the cavities on the bottom of the mold that, upon removal of the small PDMS plug, allow pneumatic or hydraulic pressure to simplify extraction of the PDMS reservoir casting to be removed from the deep, tapered mold channels that form the sides of the reservoirs. C) A photograph of the PDMS-filled mold prior to parting. D) The cast PDMS piece removed from the mold. Note that the four sets of reservoirs share a common, 1 mm thick bottom. The small, raised ring of PDMS near the edge is the overflow after an accurately measured volume of PDMS is poured into the open mold, degassed, and then displaced by the gradual lowering of the lid, with the excess PDMS being displaced into the gap between the lid and the rim. E) Device volumes. F) Port, channel, splitter, and chamber dimensions.

Fig. 3. Maturation markers of airway epithelial development. A) Cadherin-26 expression, which is associated with cell–cell junctions, epithelial integrity, and maturation, shows an increase in expression over time. B) VANGL1 is a planar cell polarity marker associated with barrier maturity that increases in expression over time as the airway epithelium develops. C) Mucins are produced by goblet cells of surface epithelium and are associated with cilia formation and a mature epithelium. D) α-Tubulin is associated with cilia development and would indicate a well-differentiated epithelium within the bronchial area. E) Barrier permeability of airway cells in a Transwell in mono- and co-culture after 28 days in culture. F) Airway barrier formation over time in a Transwell containing both endothelial and epithelial airway cells.

Fig. 4. Maturation of the co-culture airway chip. A) After 30 days, there is a functioning airway epithelial-to-vascular barrier, as shown by a significant decrease in permeability to small fluorescein (N = 9, p = .001). B) Detection of soluble ACE-2 is present after 15 days in culture in both vascular and epithelial effluent (N = 9). C) Maturation of the airway vascular endothelium shows junctional protein expression of Z0-1. D) Airway epithelium on the chip was also shown to have maturation markers previously described, including VANGL1 and mucin.

Fig. 5. Effects of Il-33 and INF cytokine exposure on ACE-2 expression and airway chip permeability. A) Effects of IL-33 and INF on ACE-2 expression were most pronounced for Hi INF, Low IL-33 + INF, and Hi IL-33 + INF (N = 4). B) Effects of IL-33 and INF on airway chip permeability, the condition which most improved ACE-2 expression without loss of barrier function, was low IL-33 + INF (N = 4). C) Table overview of low and high concentrations of cytokines.

Fig. 6. Airway chip permeability and viral load in the basal and vascular compartments in the context of SARS-CoV-2 infection. A) Permeability of the gravity airway chip was assessed by quantification of FITC-dextran levels in the basal and vascular chambers. Units were either infected with SARS-CoV-2 Italian isolate, or uninfected controls. Data are obtained as averages from N = 3 each for infected and for control samples. B) Longitudinal quantification of viral load in basal and vascular sides of the infected and control units. Data are obtained as averages from N = 3 each for all samples, and infectious titers are represented as PFU ml⁻¹. Statistical analysis was performed using two-way ANOVA with Dunnett's post-test (***p < 0.001, ns = not significant).

Fig. 7. Representative expression of three different patterns of cytokine response to SARS-CoV-2 infection in airway chips. The perfused media from the basal and the vascular sides of the units (infected [N = 3], control, [N = 3]) were collected at 24, 48, 72, 120, and 168 hours post infection. A) An early response on the basal/infected side that reduces over time. B) Robust cytokine response that is restricted to the basal side alone but maintained throughout the exposure. C) Robust cytokine response on both the basal and the vascular side. Statistical analysis was carried out using t-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.