Microphysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air–liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

COVID-19 emerged as a worldwide pandemic early in 2020, and at this writing has caused over 170 million cases and 3.7 million deaths worldwide, and almost 600,000 deaths in the United States. The rapid development of several safe and highly efficacious vaccines stands as one of the most extraordinary achievements in modern medicine, but the identification and administration of efficacious therapeutics to treat patients suffering from COVID-19 has been far less successful. A major factor limiting progress in the development of effective treatments has been a lack of suitable preclinical models for the disease, currently reliant upon various animal models and in vitro culture of immortalized cell lines. Here we report the first successful demonstration of SARS-CoV-2 infection and viral replication in a human primary cell-based organ-on-chip, leveraging a recently developed tissue culture platform known as PREDICT96. This successful demonstration of SARS-CoV-2 infection in human primary airway epithelial cells derived from a living donor represents a powerful new pathway for disease modeling and an avenue for screening therapeutic candidates in a high throughput platform.

Despite the relatively common observation of therapeutic efficacy in discovery screens with immortalized cell lines, the vast majority of drug candidates do not reach clinical development. Candidates that do move forward often fail to demonstrate efficacy when progressed from animal models to humans. This dilemma highlights the need for new drug screening technologies that can parse drug candidates early in development with regard to predicted relevance for clinical use. PREDICT96-ALI is a high-throughput organ-on-chip platform incorporating human primary airway epithelial cells in a dynamic tissue microenvironment. Here we demonstrate the utility of PREDICT96-ALI as an antiviral screening tool for SARS-CoV-2, combining the high-throughput functionality of a 96-well plate format in a high containment laboratory with the relevant biology of primary human tissue. PREDICT96-ALI resolved differential efficacy in five antiviral compounds over a range of drug doses. Complementary viral genome quantification and immunofluorescence microscopy readouts achieved high repeatability between devices and replicate plates. Importantly, results from testing the three antiviral drugs currently available to patients (nirmatrelvir, molnupiravir, and remdesivir) tracked with clinical outcomes, demonstrating the value of this technology as a prognostic drug discovery tool.