A Custom-Made Device for Reproducibly Depositing Pre-metered Doses of Nebulized Drugs on Pulmonary Cells in vitro

The deposition of pre-metered doses (i.e., defined before and not after exposition) at the air–liquid interface of viable pulmonary epithelial cells remains an important but challenging task for developing aerosol medicines. While some devices allow quantification of the deposited dose after or during the experiment, e.g., gravimetrically, there is still no generally accepted way to deposit small pre-metered doses of aerosolized drugs or pharmaceutical formulations, e.g., nanomedicines. Here, we describe a straightforward custom-made device, allowing connection to commercially available nebulizers with standard cell culture plates. Designed to tightly fit into the approximately 12-mm opening of either a 12-well Transwell® insert or a single 24-well plate, a defined dose of an aerosolized liquid can be directly deposited precisely and reproducibly (4.8% deviation) at the air–liquid interface (ALI) of pulmonary cell cultures. The deposited dose can be controlled by the volume of the nebulized solution, which may vary in a range from 20 to 200 μl. The entire nebulization-deposition maneuver is completed after 30 s and is spatially homogenous. After phosphate-buffered saline (PBS) deposition, the viability and barrier properties transepithelial electrical resistance (TEER) of human bronchial epithelial Calu-3 cells were not negatively affected. Straightforward in manufacture and use, the device enables reproducible deposition of metered doses of aerosolized drugs to study the interactions with pulmonary cell cultures grown at ALI conditions.

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Structural and functional variations in human bronchial epithelial cells cultured in air-liquid interface using different growth media

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00190.2019 ◽

2020 ◽

Vol 318 (5) ◽

pp. L1063-L1073 ◽

Cited By ~ 2

Author(s):

Clarus Leung ◽

Samuel J. Wadsworth ◽

S. Jasemine Yang ◽

Delbert R. Dorscheid

Keyword(s):

Epithelial Cells ◽

Respiratory Disease ◽

Cell Cultures ◽

Bronchial Epithelium ◽

Barrier Properties ◽

Bronchial Epithelial Cells ◽

Liquid Interface ◽

Human Bronchial Epithelial Cells ◽

Bronchial Epithelial ◽

Air Liquid Interface

The human bronchial epithelium is an important barrier tissue that is damaged or pathologically altered in various acute and chronic respiratory conditions. To represent the epithelial component of respiratory disease, it is essential to use a physiologically relevant model of this tissue. The human bronchial epithelium is a highly organized tissue consisting of a number of specialized cell types. Primary human bronchial epithelial cells (HBEC) can be differentiated into a mucociliated tissue in air-liquid interface (ALI) cultures using appropriately supplemented media under optimized growth conditions. We compared the histology, ciliary length, and function, diffusion, and barrier properties of HBEC from donors with no respiratory disease grown in two different media, PneumaCult-ALI or Bronchial Epithelial Differentiation Medium (BEDM). In the former group, HBEC have a more physiological pseudostratified morphology and mucociliary differentiation, including increased epithelial thickness, intracellular expression of airway-specific mucin protein MUC5AC, and total expression of cilia basal-body protein compared with cells from the same donor grown in the other medium. Baseline expression levels of inflammatory mediators, thymic stromal lymphopoietin (TSLP), soluble ST2, and eotaxin-3 were lower in PneumaCult-ALI. Additionally, the physiological cilia beat frequency and electrical barrier properties with transepithelial electrical resistance were significantly different between the two groups. Our study has shown that these primary cell cultures from the same donor grown in the two media possess variable structural and functional characteristics. Therefore, it is important to objectively validate primary epithelial cell cultures before experimentation to ensure they are appropriate to answer a specific scientific question.

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In well-differentiated primary human bronchial epithelial cells, TGF-β1 and TGF-β2 induce expression of furin

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00423.2020 ◽

2020 ◽

Author(s):

Michael J O'Sullivan ◽

Jennifer A Mitchel ◽

Chimwemwe Mwase ◽

Maureen McGill ◽

Phyllis Kanki ◽

...

Keyword(s):

Liquid Interface ◽

Immunofluorescence Staining ◽

Cellular Mechanisms ◽

Specific Expression ◽

Bronchial Epithelial ◽

Cellular Entry ◽

Well Differentiated ◽

Air Liquid Interface ◽

Tgf Β1

Background: The COVID-19 pandemic is an ongoing threat to public health. Since the identification of COVID-19, the disease caused by SARS-CoV-2, no drugs have been developed to specifically target SARS-CoV-2. To develop effective and safe treatment options, a better understanding of cellular mechanisms underlying SARS-CoV-2 infection is required. To fill this knowledge gap, researchers require reliable experimental systems that express the host proteins necessary for the cellular entry of SARS-CoV-2. These proteins include the viral receptor, ACE2 and the proteases TMPRSS2 and furin. A number of studies have reported cell-type specific expression of the genes encoding these molecules. However, less is known about the protein expression of these molecules. Methods: We assessed the suitability of primary human bronchial epithelial (HBE) cells maintained in air-liquid interface (ALI) as an experimental system for studying SARS-CoV-2 infection in vitro. During cellular differentiation, we measured the expression of ACE2, TMPRSS2, and furin over progressive ALI days by RT-qPCR, western blot and immunofluorescence staining. We also explored the effect of the fibrotic cytokine TGF-b on the expression of these proteins in well-differentiated HBE cells. Results/Discussion: Like ACE2, TMPRSS2 and furin proteins are localized in differentiated ciliated cells as confirmed by immunofluorescence staining. These data suggest that well-differentiated HBE cells maintained in air-liquid interface is a reliable in vitro system for investigating cellular mechanisms of SARS-CoV-2 infection. We further identified that profibrotic mediators, TGF-β1 and TGF-β2, increase the expression of furin, which is a protease required for the cellular entry of SARS-CoV-2.

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In Vitro Metabolism Of GS-424020, A Novel Mutual Prodrug Of Salmeterol And Desisobutryl-ciclesonide, BY AIR-liquid Interface HUMAN BRONCHIAL EPITHELIAL CELLS

10.1164/ajrccm-conference.2010.181.1_meetingabstracts.a6431 ◽

2010 ◽

Author(s):

Jyoti Velayudhan ◽

Joseph H. Therrien ◽

William R. Baker ◽

Joshua J. VanVeldhuizen ◽

Cliff D. Wright

Keyword(s):

Epithelial Cells ◽

Bronchial Epithelial Cells ◽

Liquid Interface ◽

In Vitro Metabolism ◽

Human Bronchial Epithelial Cells ◽

Bronchial Epithelial ◽

Air Liquid Interface ◽

Mutual Prodrug

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Agglomeration State of Titanium-Dioxide (TiO2) Nanomaterials Influences the Dose Deposition and Cytotoxic Responses in Human Bronchial Epithelial Cells at the Air-Liquid Interface

Nanomaterials ◽

10.3390/nano11123226 ◽

2021 ◽

Vol 11 (12) ◽

pp. 3226

Author(s):

Sivakumar Murugadoss ◽

Sonja Mülhopt ◽

Silvia Diabaté ◽

Manosij Ghosh ◽

Hanns-Rudolf Paur ◽

...

Keyword(s):

Titanium Dioxide ◽

Liquid Interface ◽

Glutathione Depletion ◽

In Vivo Studies ◽

Nano Tio2 ◽

Bronchial Epithelial ◽

Agglomeration State ◽

Dose Deposition ◽

Air Liquid Interface

Extensive production and use of nanomaterials (NMs), such as titanium dioxide (TiO2), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to assess the safety of TiO2 NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO2 NMs. Moreover, agglomerates are of prime importance in occupational exposure scenarios, but their toxicological relevance remains poorly understood. Therefore, the aim of this study was to investigate the potential pulmonary effects induced by TiO2 agglomerates of different sizes at the air–liquid interface (ALI), which is more realistic in terms of inhalation exposure, and compare it to results previously obtained under submerged conditions. A nano-TiO2 (17 nm) and a non-nano TiO2 (117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO2 particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO2 agglomerates at the ALI. At the end of 4h exposure, cytotoxicity, glutathione depletion, and DNA damage were evaluated. Our results indicate that dose deposition and the toxic potential in HBE cells are influenced by agglomeration and exposure via the ALI induces different cellular responses than in submerged systems. We conclude that the agglomeration state is crucial in the assessment of pulmonary effects of NMs.

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Cellular Effects in an In Vitro Human 3D Cellular Airway Model and A549/BEAS-2B In Vitro Cell Cultures Following Air Exposure to Cerium Oxide Particles at an Air–Liquid Interface

Applied In Vitro Toxicology ◽

10.1089/aivt.2015.0030 ◽

2016 ◽

Vol 2 (1) ◽

pp. 56-66 ◽

Cited By ~ 14

Author(s):

Ingeborg M. Kooter ◽

Mariska Gröllers-Mulderij ◽

Maaike Steenhof ◽

Evert Duistermaat ◽

Frederique A.A. van Acker ◽

...

Keyword(s):

Cerium Oxide ◽

Cell Cultures ◽

Liquid Interface ◽

Air Exposure ◽

Cellular Effects ◽

Oxide Particles ◽

In Vitro Cell Cultures ◽

Air Liquid Interface ◽

Airway Model

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Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00181.2012 ◽

2013 ◽

Vol 304 (7) ◽

pp. L489-L503 ◽

Cited By ~ 83

Author(s):

Carole Mathis ◽

Carine Poussin ◽

Dirk Weisensee ◽

Stephan Gebel ◽

Arnd Hengstermann ◽

...

Keyword(s):

Epithelial Cells ◽

Cigarette Smoke ◽

Bronchial Epithelium ◽

Bronchial Epithelial Cells ◽

Liquid Interface ◽

Gene Set Enrichment Analysis ◽

Bronchial Epithelial ◽

In Vitro System ◽

Air Liquid Interface

Organotypic culture of human primary bronchial epithelial cells is a useful in vitro system to study normal biological processes and lung disease mechanisms, to develop new therapies, and to assess the biological perturbations induced by environmental pollutants. Herein, we investigate whether the perturbations induced by cigarette smoke (CS) and observed in the epithelium of smokers' airways are reproducible in this in vitro system (AIR-100 tissue), which has been shown to recapitulate most of the characteristics of the human bronchial epithelium. Human AIR-100 tissues were exposed to mainstream CS for 7, 14, 21, or 28 min at the air-liquid interface, and we investigated various biological endpoints [e.g., gene expression and microRNA profiles, matrix metalloproteinase 1 (MMP-1) release] at multiple postexposure time points (0.5, 2, 4, 24, 48 h). By performing a Gene Set Enrichment Analysis, we observed a significant enrichment of human smokers' bronchial epithelium gene signatures derived from different public transcriptomics datasets in CS-exposed AIR-100 tissue. Comparison of in vitro microRNA profiles with microRNA data from healthy smokers highlighted various highly translatable microRNAs associated with inflammation or with cell cycle processes that are known to be perturbed by CS in lung tissue. We also found a dose-dependent increase of MMP-1 release by AIR-100 tissue 48 h after CS exposure in agreement with the known effect of CS on this collagenase expression in smokers' tissues. In conclusion, a similar biological perturbation than the one observed in vivo in smokers' airway epithelium could be induced after a single CS exposure of a human organotypic bronchial epithelium-like tissue culture.

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A Bioinspired in vitro Lung Model to Study Particokinetics of Nano-/Microparticles Under Cyclic Stretch and Air-Liquid Interface Conditions

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.616830 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ali Doryab ◽

Mehmet Berat Taskin ◽

Philipp Stahlhut ◽

Andreas Schröppel ◽

Sezer Orak ◽

...

Keyword(s):

Elastic Modulus ◽

Epithelial Cells ◽

Cellular Uptake ◽

Liquid Interface ◽

Alveolar Epithelial Cells ◽

Lung Model ◽

Cyclic Stretch ◽

Pulmonary Epithelial Cells ◽

Air Liquid Interface

Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m2) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic InVItroCell-stretch (CIVIC) “breathing” lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 μm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o−) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35% PCL and 6.34% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung.

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