Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens <em>In vitro</em>

Abstract

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult 1 , pharmacological compounds [2][3][4][5][6] , and bacterial 7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome -associated coronavirus [10][11][12][13][14] . Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE 15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating transepithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments 17,18 . Once TEER plateaus at or above 1,000 Ω×cm 2 , Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens. Video LinkThe video component of this article can be found at http://www.jove.com/video/50157/ Protocol Culturing Calu-3 Cells for Use in Transwell CulturesSafety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique.

Glycosylation changes in the globular head of H3N2 influenza hemagglutinin modulate receptor binding without affecting virus virulence

“…We also compared virus replication in Calu-3 cells grown in liquid-covered culture (LCC), which does not produce mucin, or in air-interface culture (AIC), which does produce mucin and most closely mimics the conditions of RT, at temperatures reflective of the proximal (32 °C) and distal (37 °C) airways. The Calu-3 cell line has features of differentiated, functional human airway epithelial cells31 (including forming polarized layers with apical and basolateral surfaces and secreting mucin), expresses SA receptors preferred by human H3N2 viruses32, and is a well-established human respiratory epithelial cell model for IAV infection3233.…”

Endothelial cells' biophysical, biochemical, and chromosomal aberrancies in high‐glucose condition within the diabetic range

“…To monitor the effect of HGC on endothelial monolayer integrity, we simultaneously added 2 × 10 5 cells and 600 μL of DMEM‐F12 without cell and any supplements into the apical and basolateral compartments of a 24‐well Transwell system, respectively . To measure the electrical resistance through a monolayer of ECs, we placed multimeter volt‐ohmmeter electrodes (model: Victor 86D, Xiamen Wellzion Electronics) either in the basolateral or in the apical compartment.…”

“…The assay was recorded for a total of 3 measurements per well. Finally, unit area resistance was calculated using following equations (Equations 1 and 2): where the effective membrane area stands for 0.33 cm 2 for 24‐well Transwell inserts.…”

Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions

“…Comparative profiling of spores in the commonly used immortalized lung epithelial cells A549 and Calu‐3 was also carried out (Foster et al . ; Harcourt and Haynes ). It was our intent that by assessing spore exposure outcomes within various lung cell lines under undifferentiated and differentiated conditions, we could better define parameters relating to lung spore exposure in vitro that are presently difficult to measure in vivo .…”