High-Throughput Human Primary Cell-Based Airway Model for Evaluating Influenza, Coronavirus, or other Respiratory Virusesin vitro

Abstract

Influenza and other respiratory viruses represent a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as the current COVID-19 crisis. One of the greatest barriers to the development of effective therapeutic agents to treat influenza, coronaviruses, and many other infections of the respiratory tract is the absence of a robust preclinical model. Preclinical studies currently rely on high-throughput, low-fidelity in vitro screening with cell lines and/or low-throughput animal models that often provide a poor correlation to human clinical responses. Here, we introduce a human primary airway epithelial cell-based model integrated into a highthroughput platform where tissues are cultured at an air-liquid interface (PREDICT96-ALI). We present results on the application of this platform to influenza and coronavirus infections, providing multiple readouts capable of evaluating viral infection kinetics and potentially the efficacy of therapeutic agents in an in vitro system. Several strains of influenza A virus are shown to successfully infect the human primary cell-based airway tissue cultured at an air-liquid interface (ALI), and as a proof-of-concept, the effect of the antiviral oseltamivir on one strain of Influenza A is evaluated. Human coronaviruses NL63 (HCoV-NL63) and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for protein priming, and we confirm expression of both in our ALI model. We also demonstrate coronavirus infection in this system with HCoV-NL63, observing sufficient viral propagation over 96 hours post-infection to indicate successful infection of the primary cell-based model. This new capability has the potential to address a gap in the rapid assessment of therapeutic efficacy of various small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

Is it Time for Reviewer 3 to Request Human Organ Chip Experiments Instead of Animal Validation Studies?

“…[20,21] Microfluidic Airway Chips are now being used to model human lung infection by SARS-CoV-2 virus in vitro, and to repurpose existing FDAapproved drugs as potential COVID-19 therapeutics. [21,22] Importantly, it is extremely difficult to develop animal models of viral infection, and none replicate human host responses as well as these Organ Chip models that incorporate highly differentiated human lung cells grown under an ALI while exposed to dynamic fluid flow at their base.…”

SARS-CoV-2 Viral Replication in a High Throughput Human Primary Epithelial Airway Organ Model

“…The PREDICT96 organ-on-chip platform consists of a microfluidic culture plate with 96 individual devices and a perfusion system driven by 192 microfluidic pumps integrated into the plate lid 17 . As described in our previous work 13,[16][17][18] , in order to establish recirculating flow in the microchannels, we incorporate a self-priming micropump array into the lid that serves as the fluidic interface with the culture plate via stainless steel tubes. The PREDICT96 pumping system has 192 individual pneumatically-actuated micropumps embedded in the plate lid: two per culture device, one for the chamber above the membrane and one for below.…”

“…Donor cells reported in this manuscript were derived from donor DH01: 28 years, female, Asian (non-Hispanic). Cells were cultured in SAGM + 4i media 13 , and passaged using Accutase (Sigma). After 4 passages, cells were cryopreserved in Cryostor 10 (STEMCELL Technologies).…”

“…In response to these challenges, the field of organs-on-chips has turned intense attention toward modeling of respiratory pathogens such as influenza and SARS-CoV-2, and a number of groups have reported results relevant to the effort to establish therapeutic screening tools for these diseases [10][11][12][13] . These platforms utilize human primary alveolar or airway epithelial cells in microfluidic organ models designed to closely mimic not only the relevant cell biology but other key aspects of the in vivo microenvironment.…”

“…Here we report the first demonstration of SARS-CoV-2 infection and viral replication in a human primary cell-based airway-on-a-chip organ model. We utilized the PREDICT96-ALI 13 system that has previously been demonstrated as a platform for the study of infection human airway tissue models with influenza virus and hCoV-NL63, in collaboration with the University of Massachusetts Medical School under the DARPA PREPARE program. Unlike earlier studies, this work investigates the inoculation of live SARS-CoV-2 virus into human primary airway epithelial cells in a BSL-3 environment, an important step forward in the arsenal of preclinical tools for modeling COVID-19 infection.…”