Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics

Longlong Si; Haiqing Bai; Melissa Rodas; Wuji Cao; Crystal Yur Oh; Amanda Jiang; Atiq Nurani; Danni Y Zhu; Girija Goyal; Sarah Gilpin; Rachelle Prantil-Baun; Donald E. Ingber

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.13.039917 doi: bioRxiv preprint responses, mucociliary clearance, host-microbiome interactions, and other relevant human organ-level physiological behaviors in vitro 4-8,37,38,47 , perhaps the greatest value of human Organ Chips is that they also could be used to study human-relevant immune responses to infectious virus and disease pathogenesis, which are difficult to replicate in other in vitro or in vivo models. We therefore hope to move this technology into BSL3certified laboratories in the near future so that similar studies can be carried out with native SARS-CoV2 over the coming months. The existing approved drugs found to be active in both established cell lines and human Organ Chips described here potentially could be useful as prophylactics or therapeutics against SARS-CoV-2 and influenza infections, and thus also might be considered for further evaluation in human clinical trials. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.13.039917 doi: bioRxiv preprint coating. The channels were then washed sequentially with ER2 buffer and PBS. The porous membranes were coated on both sides with collagen type IV from human placenta (0.5 mg/mL in water; Sigma-Aldrich) at room temperature overnight. The solution was then aspirated from the chip, which was then used for seeding cells. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.13.039917 doi: bioRxiv preprint (StemCell) supplemented with 0.1% VEGF, 0.01% EGF, and 1mM CaCl2 from an Endothelial Cell Medium Kit (Cell Biological, M1168) through the bottom vascular channel. The chips were cultured in an incubator containing 5% CO2 and 16-18% O2 at 85-95% humidity, and the apical surface of the epithelium was rinsed once weekly with PBS to remove cellular debris and mucus. Highly differentiated human airway structures and functions can be maintained in the human lung Airway chip for more than 2 months. Immunofluorescence microscopy. Cells were washed with PBS through the apical and basal channels, fixed with 4% paraformaldehyde (Alfa Aesar) for 20-25 min, and then washed with PBS before being stored at 4 o C. Fixed tissues were permeabilized on-chip with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, exposed to PBS with 10% goat serum (Life Technologies) and 0.1% Triton X-100 for 30 min at room temperature, and then incubated with primary antibodies (Supplementary Table 2 ) diluted in incubation buffer (PBS with 1% goat serum and 0.1% Triton X-100) overnight at 4 o C, followed by incubation with corresponding secondary antibodies (Supplementary Table 2 ) for 1 h at room temperature; nuclei were counterstained with DAPI (Invitrogen) after secondary antibody staining. Fluorescence imaging was carried out using a confocal laser-scanning microscope (SP5 X MP DMI-6000, Germany) and image processing was done using Imaris software (Bitplane, Switzerland).