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

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.

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CFTR is required for maximal transepithelial liquid transport in pig alveolar epithelia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00116.2012 ◽

2012 ◽

Vol 303 (2) ◽

pp. L152-L160 ◽

Cited By ~ 21

Author(s):

Xiaopeng Li ◽

Alejandro P. Comellas ◽

Philip H. Karp ◽

Sarah E. Ernst ◽

Thomas O. Moninger ◽

...

Keyword(s):

Cystic Fibrosis ◽

Liquid Interface ◽

Alveolar Epithelial Cells ◽

Limiting Factor ◽

Apical Surface ◽

Liquid Transport ◽

Liquid Height ◽

Liquid Absorption ◽

Air Liquid Interface ◽

Alveolar Liquid

A balance between alveolar liquid absorption and secretion is critical for maintaining optimal alveolar subphase liquid height and facilitating gas exchange in the alveolar space. However, the role of cystic fibrosis transmembrane regulator protein (CFTR) in this homeostatic process has remained elusive. Using a newly developed porcine model of cystic fibrosis, in which CFTR is absent, we investigated ion transport properties and alveolar liquid transport in isolated type II alveolar epithelial cells (T2AECs) cultured at the air-liquid interface. CFTR was distributed exclusively to the apical surface of cultured T2AECs. Alveolar epithelia from CFTR−/− pigs failed to increase liquid absorption in response to agents that increase cAMP, whereas cAMP-stimulated liquid absorption in CFTR+/− epithelia was similar to that in CFTR+/+ epithelia. Expression of recombinant CFTR restored stimulated liquid absorption in CFTR−/− T2AECs but had no effect on CFTR+/+ epithelia. In ex vivo studies of nonperfused lungs, stimulated liquid absorption was defective in CFTR−/− alveolar epithelia but similar between CFTR+/+ and CFTR+/− epithelia. When epithelia were studied at the air-liquid interface, elevating cAMP levels increased subphase liquid height in CFTR+/+ but not in CFTR−/− T2AECs. Our findings demonstrate that CFTR is required for maximal liquid absorption under cAMP stimulation, but it is not the rate-limiting factor. Furthermore, our data define a role for CFTR in liquid secretion by T2AECs. These insights may help to develop new treatment strategies for pulmonary edema and respiratory distress syndrome, diseases in which lung liquid transport is disrupted.

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Antioxidant properties of guinea pig tracheal epithelial cells in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.4.l397 ◽

1994 ◽

Vol 266 (4) ◽

pp. L397-L404 ◽

Cited By ~ 1

Author(s):

L. A. Cohn ◽

V. L. Kinnula ◽

K. B. Adler

Keyword(s):

Guinea Pig ◽

Matrix Material ◽

Antioxidant Properties ◽

Liquid Interface ◽

Apical Surface ◽

Scavenging Activity ◽

Tracheal Epithelial Cells ◽

Tracheal Epithelial ◽

Air Liquid Interface

Guinea pig tracheal epithelial (GPTE) cells in primary air/liquid interface culture were exposed to H2O2, and the rate of H2O2 consumption by apical and basolateral surfaces was measured. GPTE cells had potent H2O2 scavenging ability, with faster consumption of H2O2 from the apical surface. Inhibition of catalase (Cat) with sodium azide (NaAz) significantly attenuated the ability of GPTE cells to remove higher concentrations of H2O2. Depletion of reduced glutathione, the substrate for glutathione peroxidase (GPO), with DL-buthionine-[S,R]-sulfoximine (BSO) did not affect consumption of H2O2. Dissolution of mucus from the cells reduced scavenging activity of the cultures and basement membrane/extracellular matrix material (BM/ECM) deposited by the cells demonstrated significant H2O2-scavenging activity. The results suggest that GPTE cells retain antioxidant capability in vitro when cultured in an air/liquid interface. This capacity to scavenge H2O2 appears to rely on Cat, as well as on mucus and BM/ECM material. However, a significant amount of H2O2 scavenging appears to depend on other, yet unidentified, antioxidant system(s).

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Proteomics-Based Identification of Proteins Secreted in Apical Surface Fluid of Squamous Metaplastic Human Tracheobronchial Epithelial Cells Cultured by Three-Dimensional Organotypic Air-Liquid Interface Method

Cancer Research ◽

10.1158/0008-5472.can-06-2783 ◽

2007 ◽

Vol 67 (14) ◽

pp. 6565-6573 ◽

Cited By ~ 20

Author(s):

Seung-Wook Kim ◽

Kyounga Cheon ◽

Chang-Hoon Kim ◽

Joo-Heon Yoon ◽

David H. Hawke ◽

...

Keyword(s):

Epithelial Cells ◽

Three Dimensional ◽

Liquid Interface ◽

Apical Surface ◽

Air Liquid Interface ◽

Cells Cultured

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Temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface

10.1101/197814 ◽

2017 ◽

Cited By ~ 1

Author(s):

Daniel Cozens ◽

Erin Sutherland ◽

Francesco Marchesi ◽

Geraldine Taylor ◽

Catherine Berry ◽

...

Keyword(s):

Epithelial Cells ◽

Ex Vivo ◽

Animal Health ◽

Bronchial Epithelium ◽

Airway Epithelial Cells ◽

Liquid Interface ◽

Mucosal Barrier ◽

Basal Cells ◽

Airway Epithelial ◽

Air Liquid Interface

AbstractThe respiratory epithelium is exposed to assault by toxins and pathogens through the process of inhalation, which has numerous implications on both human and animal health. As such, there is a need to develop and characterise anin vitromodel of the airway epithelium to study respiratory pathologies during infection or toxicology experiments. This has been achieved by growing airway epithelial cells at an air-liquid interface (ALI). Characterisation of ALI models are not well-defined for airway epithelial cells derived from non-human species. In this study we have fully characterised a bovine airway epithelial cell models (AECM) grown at an ALI in relation toex vivotissue. The morphology of the model was monitored at three day intervals, to identify the time-period at which the culture was optimally differentiated. The model was shown to be fully-differentiated by day 21 post-ALI. The culture formed a stereotypical pseudostratified, columnar epithelium containing the major cell types of the bronchial epithelium (ciliated-, goblet- and basal cells). Once fully differentiated the bovine AECM displayed both barrier function, through the formation of tight-junctions, and active mucociliary clearance, important properties of the mucosal barrier. The bovine bronchial epithelial cells remained stable for three weeks, with no evidence of deterioration or dedifferentiation. The window in which the model displayed full differentiation was determined to be between day 21-42 post-ALI. Through comparison withex vivotissue derived from donor animals, our bovine AECM was shown to be highly representative of thein vivobovine bronchial epithelium and can be utilised in the study of respiratory pathologies.

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