Murine Tracheal and Nasal Septal Epithelium for Air–Liquid Interface Cultures: A Comparative Study

2007 ◽  
Vol 21 (5) ◽  
pp. 533-537 ◽  
Author(s):  
Bradford A. Woodworth ◽  
Marcelo B. Antunes ◽  
Geeta Bhargave ◽  
James N. Palmer ◽  
Noam A. Cohen
Keyword(s):  
Epithelial Cells ◽  
Respiratory Epithelium ◽  
Liquid Interface ◽  
Respiratory Epithelial Cells ◽  
Cell Counts ◽  
Total Cell Population ◽  
Respiratory Epithelial ◽  
Air Liquid Interface ◽  
Ciliated Epithelial Cells

Background Air–liquid interface cultures using murine tracheal respiratory epithelium have revolutionized the in vitro study of airway diseases. However, these cultures often are impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. The ability to study ciliary physiology in vitro is of utmost importance in the research of chronic rhinosinusitis (CRS). Our hypothesis is that the murine nasal septum is a better source of ciliated respiratory epithelium to develop respiratory epithelial air–liquid interface models. Methods Nasal septa and tracheas were harvested from 10 BALB/c mice. The nasal septa were harvested by using a simple and straightforward novel technique. Scanning electron microscopy was performed on all specimens. Cell counts of ciliated respiratory epithelial cells were performed at one standard magnification (1535×). Comparative analysis of proximal and distal trachea, midanterior and midposterior nasal septal epithelium, was performed. Results Independent cell counts revealed highly significant differences in the proportion of cell populations (p < 0.00001). Ciliated cell counts for the trachea (106.9 ± 28) were an average of 38.7% of the total cell population. Nasal septal ciliated epithelial cells (277.5 ± 16) comprised 90.1% of the total cell population. Conclusion To increase the yield of respiratory epithelial cells harvested from mice, we have found that the nasal septum is a far superior source when compared with the trachea. The greater surface area and increased concentration of ciliated epithelial cells has the potential to provide an eightfold increase in epithelial cells for the development of air–liquid interface cultures.

scholarly journals Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface

2020 ◽  
Vol 318 (6) ◽  
pp. L1158-L1164
Author(s):  
Emily Mavin ◽  
Bernard Verdon ◽  
Sean Carrie ◽  
Vinciane Saint-Criq ◽  
Jason Powell ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Real Time ◽  
Aerobic Glycolysis ◽  
Airway Epithelial Cells ◽  
Respiratory Epithelium ◽  
Liquid Interface ◽  
Human Nasal Epithelial Cells ◽  
Respiratory Epithelial ◽  
Air Liquid Interface

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

scholarly journals Viral Coinfection Replaces Effects of Suilysin onStreptococcus suisAdherence to and Invasion of Respiratory Epithelial Cells Grown under Air-Liquid Interface Conditions

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Fandan Meng ◽  
Jie Tong ◽  
Désirée Vötsch ◽  
Ju-Yi Peng ◽  
Xuehui Cai ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Bacterial Infection ◽  
Liquid Interface ◽  
Influenza Viruses ◽  
Striking Difference ◽  
Respiratory Epithelial Cells ◽  
Content Type ◽  
Secondary Bacterial Infection ◽  
Respiratory Epithelial ◽  
Air Liquid Interface

ABSTRACTStreptococcus suisis an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis ofS. suisinfections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence ofS. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin ofS. suiscontributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt)S. suisstrain and a suilysin-negativeS. suismutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wtS. suisstrain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negativeS. suis.

scholarly journals Receptor analogs and monoclonal antibodies that inhibit adherence of Bordetella pertussis to human ciliated respiratory epithelial cells.

1988 ◽  
Vol 168 (1) ◽  
pp. 267-277 ◽  
Author(s):  
E Tuomanen ◽  
H Towbin ◽  
G Rosenfelder ◽  
D Braun ◽  
G Larson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Ciliated Cells ◽  
Respiratory Epithelial Cells ◽  
Respiratory Cilia ◽  
Respiratory Epithelial ◽  
Ciliated Epithelial Cells ◽  
Receptor Antibodies

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough. To explore the development of agents that could interrupt adherence, the structure of the receptor on the ciliary surface was investigated. Using an in vitro adherence assay to human ciliated epithelial cells, galactose, lactose, and complex carbohydrates containing lactose eliminated adherence when preincubated with the bacteria. 10(-2) M galactose eluted adherent bacteria from cilia. B. pertussis and its two purified adhesins bound specifically to natural lactose-containing glycolipids in a TLC assay. mAbs to eukaryotic glycoconjugates with specificity for substituted galactose-glucose moieties blocked adherence when preincubated with ciliated cells. The carbohydrates that serve as receptors for B. pertussis on human cilia are galactose-glucose-containing glycolipids. Receptor analogs and anti-receptor antibodies effectively block adherence of B. pertussis to cilia and thus should be considered candidates for therapeutic intervention against disease.

scholarly journals In Vitro Studies of Mechanisms of Lung Injury in the Rodent

1991 ◽  
Vol 19 (4_part_1) ◽  
pp. 419-427 ◽  
Author(s):  
Leah A. Cohn ◽  
Kenneth B. Adler
Keyword(s):  
Epithelial Cells ◽  
Cell Types ◽  
Lung Cells ◽  
Serum Free Medium ◽  
Collagen Gels ◽  
Respiratory Epithelial Cells ◽  
Respiratory Epithelial ◽  
Air Liquid Interface

In order to better define the responses of lung cells to potentially pathogenic insults, primary cell cultures of dissociated respiratory epithelial cells have been established. These epithelial cells have been obtained from various areas of the respiratory tract ranging from the trachea to the alveolus and the cultures have been demonstrated to mimic the differentiated state of these cell types as observed in situ. Several procedures which enhance the differentiated state have been evaluated, which include maintenance on more physiologically-relevant substrata, such as collagen gels, use of defined serum-free medium and use of air/liquid interface systems. These approaches have allowed intracellular responses of respiratory epithelium to toxic insult to be better defined.

scholarly journals Centriolar Satellites

1999 ◽  
Vol 147 (5) ◽  
pp. 969-980 ◽  
Author(s):  
Akiharu Kubo ◽  
Hiroyuki Sasaki ◽  
Akiko Yuba-Kubo ◽  
Shoichiro Tsukita ◽  
Nobuyuki Shiina
Keyword(s):  
Epithelial Cells ◽  
Live Cells ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Respiratory Epithelial Cells ◽  
Dense Granules ◽  
Pericentriolar Material ◽  
Respiratory Epithelial ◽  
Ciliated Epithelial Cells

We identified Xenopus pericentriolar material-1 (PCM-1), which had been reported to constitute pericentriolar material, cloned its cDNA, and generated a specific pAb against this molecule. Immunolabeling revealed that PCM-1 was not a pericentriolar material protein, but a specific component of centriolar satellites, morphologically characterized as electron-dense granules, ∼70–100 nm in diameter, scattered around centrosomes. Using a GFP fusion protein with PCM-1, we found that PCM-1–containing centriolar satellites moved along microtubules toward their minus ends, i.e., toward centrosomes, in live cells, as well as in vitro reconstituted asters. These findings defined centriolar satellites at the molecular level, and explained their pericentriolar localization. Next, to understand the relationship between centriolar satellites and centriolar replication, we examined the expression and subcellular localization of PCM-1 in ciliated epithelial cells during ciliogenesis. When ciliogenesis was induced in mouse nasal respiratory epithelial cells, PCM-1 immunofluorescence was markedly elevated at the apical cytoplasm. At the electron microscopic level, anti–PCM-1 pAb exclusively labeled fibrous granules, but not deuterosomes, both of which have been suggested to play central roles in centriolar replication in ciliogenesis. These findings suggested that centriolar satellites and fibrous granules are identical novel nonmembranous organelles containing PCM-1, which may play some important role(s) in centriolar replication.

scholarly journals Mycoplasma pneumoniae carriage evades induction of protective mucosal antibodies

2021 ◽  
pp. 2100129
Author(s):  
Ruben Cornelis Anthonie de Groot ◽  
Silvia Cristina Estevão ◽  
Patrick Michael Meyer Sauteur ◽  
Aditya Perkasa ◽  
Theo Hoogenboezem ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mycoplasma Pneumoniae ◽  
Community Acquired Pneumonia ◽  
Respiratory Epithelial Cells ◽  
Specific Antibodies ◽  
Asymptomatic Carriage ◽  
Nasal Secretions ◽  
Respiratory Epithelial ◽  
Nested Case Control

Mycoplasma pneumoniae is the most common bacterial cause of pneumonia in children hospitalised for community-acquired pneumonia. Prevention of infection by vaccines may be an important strategy in the presence of emerging macrolide resistant M. pneumoniae. However, knowledge of immune responses to M. pneumoniae is limited, complicating vaccine design. We therefore studied the antibody response during M. pneumoniae infection and asymptomatic carriage.In a nested case-control study (n=80) of M. pneumoniae carriers and matched controls we observed that carriage by M. pneumoniae does not lead to a rise in either mucosal or systemic M. pneumoniae-specific antibodies, even after months of persistent carriage. We replicated this finding in a second cohort (n=69) and also found that during M. pneumoniae community-acquired pneumonia, mucosal levels of M. pneumoniae-specific IgA and IgG did increase significantly. In vitro adhesion assays revealed that high levels of M. pneumoniae-specific antibodies in nasal secretions of paediatric patients prevented the adhesion of M. pneumoniae to respiratory epithelial cells.In conclusion, our study demonstrates that M. pneumoniae-specific mucosal antibodies protect against bacterial adhesion to respiratory epithelial cells and are induced only during M. pneumoniae infection and not during asymptomatic carriage. This is strikingly different from carriage with bacteria such as Streptococcus pneumoniae where mucosal antibodies are induced by bacterial carriage.

In Vitro High-Content Imaging-Based Phenotypic Analysis of Bronchial 3D Organotypic Air–Liquid Interface Cultures

2020 ◽  
Vol 25 (3) ◽  
pp. 247-252
Author(s):  
Diego Marescotti ◽  
David Bovard ◽  
Moran Morelli ◽  
Antonin Sandoz ◽  
Karsta Luettich ◽  
...  
Keyword(s):  
Biological Effects ◽  
Liquid Interface ◽  
Phenotypic Analysis ◽  
Cell Hyperplasia ◽  
High Content Imaging ◽  
Differentiation Status ◽  
Third Dimension ◽  
Air Liquid Interface ◽  
Ciliated Epithelial Cells

High-content imaging (HCI) is a powerful method for quantifying biological effects in vitro. Historically, HCI has been applied to adherent cells growing in monolayers. With the advent of confocal versions of HCI devices, researchers now have the option of performing analyses on 3D cell cultures. However, some obstacles remain in integrating the third dimension, such as limited light penetration and less sophisticated image analysis. Here, we report the development of an HCI technique for imaging human bronchial 3D organotypic air–liquid interface (ALI) cultures (hBR-ALI). In this method, we monitored differentiation status through HCI evaluation markers representative of ciliated epithelial cells and goblet cells (Muc5AC [mucin 5AC]). As a second use case for demonstrating the utility of this technique, we induced goblet cell hyperplasia in hBR-ALI by using interleukin (IL)-13. Our results demonstrate the utility of the HCI technique for imaging hBR-ALI grown on Transwell inserts. This technique may be expanded to other cell culture systems, such as skin epithelia and 3D intestinal systems.

Sign in / Sign up

Export Citation Format

Share Document