Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection

Abstract

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air–liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

Organoid Models of SARS-CoV-2 Infection: What Have We Learned about COVID-19?

“…In the respiratory tract, the ACE2 receptor is preferentially expressed on the apical surface of epithelial cells. Both SARS-CoV and SARS-CoV-2 infect by binding apically distributed ACE2 and during their egress from infected cells, are also released from the apical surface of respiratory cells [29][30][31][32]. This is significant as our understanding of viral entry, release and neutralization may be guided very differently by studying these events in polarized, compared to non-polarized, respiratory epithelial cells.…”

“…ALI-differentiation medium is refreshed in the basal chamber over 3-4 weeks to generate the pseudostratified tissue. Matrix embedded organoids are similarly seeded in an expansion phase to form cysts, and then changed to an organoid differentiation medium for 3-4 weeks [8,32]. HAE organoids are comprised of several cell types, including basal cells, ciliated cells with apical beating cilia and goblet cells secreting mucous onto the apical surface.…”

“…Early expression studies either did not detect or detected low levels [87] or did not report [85] ACE2 protein expression in respiratory tissues, despite robust protein expression reported in other human tissues such as the intestine analyzed in parallel [85,87]. More recent studies have confirmed ACE2 protein expression and SARS-CoV-2 infections in respiratory epithelia, identifying the differentiated ciliated cells as the dominant cell type infected [21,29,32,88,89] (reviewed extensively elsewhere, e.g., [90][91][92]).…”

“…Organoids 2022, 1, FOR PEER REVIEW 7 [87] or did not report [85] ACE2 protein expression in respiratory tissues, despite robust protein expression reported in other human tissues such as the intestine analyzed in parallel [85,87]. More recent studies have confirmed ACE2 protein expression and SARS-CoV-2 infections in respiratory epithelia, identifying the differentiated ciliated cells as the dominant cell type infected [21,29,32,88,89] (reviewed extensively elsewhere, e.g., [90][91][92]). With the urgency to discover and evaluate therapies of severe respiratory damage in COVID-19, researchers have sought to establish human alveolar models of SARS-CoV-2 infection (Figure 4).…”

“…6.3. Accurate Pre-Clinical Models of SARS-CoV-2 Transmission and Respiratory Disease ALI-HNE and matrix-embedded HNE organoids are proving to be excellent models of SARS-CoV-2 variant transmissibility [32,227,228]. Infection of ALI-HNE with an ancestral (Wuhan) clinical isolate [229] did not cause overt cytopathic effects, despite yielding a high titer virus from the apical surface.…”