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

Download Full-text

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.

Download Full-text

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

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.643491 ◽

2021 ◽

Vol 9 ◽

Author(s):

Justus C. Horstmann ◽

Chelsea R. Thorn ◽

Patrick Carius ◽

Florian Graef ◽

Xabier Murgia ◽

...

Keyword(s):

Cell Cultures ◽

Pharmaceutical Formulations ◽

Barrier Properties ◽

Liquid Interface ◽

Bronchial Epithelial ◽

Pulmonary Epithelial Cells ◽

Custom Made ◽

Air Liquid Interface ◽

Phosphate Buffered Saline

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.

Download Full-text

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

Download Full-text

Parametric Optimization of an Air–Liquid Interface System for Flow-Through Inhalation Exposure to Nanoparticles: Assessing Dosimetry and Intracellular Uptake of CeO2 Nanoparticles

Nanomaterials ◽

10.3390/nano10122369 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2369 ◽

Cited By ~ 1

Author(s):

Lars Leibrock ◽

Harald Jungnickel ◽

Jutta Tentschert ◽

Aaron Katz ◽

Blaza Toman ◽

...

Keyword(s):

Co2 Concentration ◽

Liquid Interface ◽

In Vivo Studies ◽

Inhalation Toxicity ◽

Exposure System ◽

Inhalation Study ◽

Flow Through ◽

Air Liquid Interface

Air–liquid interface (ALI) systems have been widely used in recent years to investigate the inhalation toxicity of many gaseous compounds, chemicals, and nanomaterials and represent an emerging and promising in vitro method to supplement in vivo studies. ALI exposure reflects the physiological conditions of the deep lung more closely to subacute in vivo inhalation scenarios compared to submerged exposure. The comparability of the toxicological results obtained from in vivo and in vitro inhalation data is still challenging. The robustness of ALI exposure scenarios is not yet well understood, but critical for the potential standardization of these methods. We report a cause-and-effect (C&E) analysis of a flow through ALI exposure system. The influence of five different instrumental and physiological parameters affecting cell viability and exposure parameters of a human lung cell line in vitro (exposure duration, relative humidity, temperature, CO2 concentration and flow rate) was investigated. After exposing lung epithelia cells to a CeO2 nanoparticle (NP) aerosol, intracellular CeO2 concentrations reached values similar to those found in a recent subacute rat inhalation study in vivo. This is the first study showing that the NP concentration reached in vitro using a flow through ALI system were the same as those in an in vivo study.

Download Full-text

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.

Download Full-text