Biomimetic Alveolus-on-a-Chip for SARS-CoV-2 Infection Recapitulation

Abstract

SARS-CoV-2 has caused a severe pneumonia pandemic worldwide with high morbidity and mortality. How to develop a preclinical model for recapitulating SARS-CoV-2 pathogenesis is still urgent and essential for the control of the pandemic. Here, we have established a 3D biomimetic alveolus-on-a-chip with mechanical strain and extracellular matrix taken into consideration. We have validated that the alveolus-on-a-chip is capable of recapitulating key physiological characteristics of human alveolar units, which lays a fundamental basis for viral infection studies at the organ level. Using virus-analogous chemicals and pseudovirus, we have explored virus pathogenesis and blocking ability of antibodies during viral infection. This work provides a favorable platform for SARS-CoV-2-related researches and has a great potential for physiology and pathophysiology studies of the human lung at the organ level in vitro.

Figure 1

The 3D alveolus-on-a-chip platform. (a) Schematics of the alveolus and alveolar-capillary barrier in vivo. The alveolar unit has a key feature of epithelial cell/extracellular matrix/endothelial cell structure. (b) Design and structure of the microfluidic chip. It contains an epithelial cell (ihAEpiC) culture channel (cyclic air mechanical strain can be induced), endothelial cell (HUVEC) culture channel (continuous medium flow can be induced), and central collagen I gel channel.

Figure 2

(a) HUVEC and ihAEpiC were seeded onto Matrigel and collagen I gel and normally cultured for 1 d. Scale bar is 200 μm. (b) The average cell adhesion area of ihAEpiC and HUVEC on the side of collagen gel in the microchip. Error bars mean standard deviation, and each dot means one independent experiment. For the static cell culture mode, chips were normally cultured in a CO₂ incubator and cell medium was refreshed manually every 12 h. For the dynamic cell culture mode, chips were cultured at the same experimental condition but with a flow injection pump and cell medium was continuously refreshed with an injection pump at a flow rate of 20 μL/h, as shown in Figure S4 (Supporting Information). (c) The live and dead cell staining. Green fluorescence means live cells stained with calcein AM. Red fluorescence means dead cells stained with PI. (d) Result of E-cadherin and VE-cadherin immunofluorescent stain. The enlarged images show the observations under a confocal microscope with a water immersion objective. Scale bar is 50 μm.

Figure 3

(a) Alveolar epithelial cells with/without cyclic air stimulation were stained with FM1-43 dye and viewed under a confocal microscope. Enlarged images with a large zoom magnification were shown to provide a single intact epithelial cell staining morphology. Scale bar is 50 μm for zoom 1 and 5 μm for zoom 7. (b) Actin was immunofluorescently stained with TRITC-phalloidin for endothelial cells stimulated with continuous medium flow at a flow rate of 60 μL/h for 0 h and 2 h. Vector orientation is visualized based on the orientation of the actin image in the vector field; the orientation distribution is computed based on the structure tensor for each pixel and visualized in a color map in the HSB mode (hue is orientation, saturation is coherency, and brightness is the source image) in an HSB color-coded map. Scale bar is 50 μm.

Figure 4

(a) ROS generation characterization before and after poly(I:C) treatment for ihAEpiC (scale bar, 50 μm) and HUVEC (scale bar, 100 μm). Cytokines secreted by (b) ihAEpiC and (c) HUVEC before and after poly(I:C) treatment. (d) Images of U937 cells adhering to HUVEC and number calculation of adhered U937 cells within six random areas in the microchip. Scale bar is 100 μm.

Figure 5

(a) Pseudoviral infection with different viral concentrations in ihAEpiC and HUVEC. (b) The influence of the SARS-CoV-2 Spike-RBD monoclonal antibody on viral infection. Scale bar is 50 μm.