multicellular-human-alveolar-model-composed-epithelial-cells-primary

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

University of Fribourg

There are justifiable health concerns regarding the potential adverse effects associated with human exposure to volcanic ash (VA) particles, especially when considering communities living in urban areas already exposed to heightened air pollution. The aim of this study was, therefore, to gain an imperative, first understanding of the biological impacts of respirable VA when exposed concomitantly with diesel particles.

Combined exposure of diesel exhaust particles and respirable Soufrière Hills volcanic ash causes a (pro-)inflammatory response in an in vitro multicellular epithelial tissue barrier model.

Although acute pulmonary toxicity of carbon nanotubes (CNTs) has been extensively investigated, the knowledge of potential health effects following chronic occupational exposure is currently limited and based only upon in vivo approaches. Our aim was to realistically mimic subchronic inhalation of multiwalled CNTs (MWCNTs) in vitro, using the air-liquid interface cell exposure (ALICE) system for aerosol exposures on reconstituted human bronchial tissue from healthy and asthmatic donors. The reliability and sensitivity of the system were validated using crystalline quartz (DQ12), which elicited an increased (pro-)inflammatory response, as reported in vivo. At the administrated MWCNT doses relevant to human occupational lifetime exposure (10 μg/cm for 5 weeks of repeated exposures/5 days per week) elevated cilia beating frequency (in both epithelial cultures), and mucociliary clearance (in asthmatic cells only) occurred, whereas no cytotoxic reactions or morphological changes were observed. However, chronic MWCNT exposure did induce an evident (pro-)inflammatory and oxidative stress response in both healthy and asthmatic cells. The latter revealed stronger and more durable long-term effects compared to healthy cells, indicating that individuals with asthma may be more susceptible to adverse effects from chronic MWCNT exposure. Our results highlight the power of occupationally relevant subchronic exposures on human in vitro models in nanosafety hazard assessment.

Human Asthmatic Bronchial Cells Are More Susceptible to Subchronic Repeated Exposures of Aerosolized Carbon Nanotubes At Occupationally Relevant Doses Than Healthy Cells.

LiCoO is one of the most used cathode materials in Li-ion batteries. Its conventional synthesis requires high temperature (>800 °C) and long heating time (>24 h) to obtain the micronscale rhombohedral layered high-temperature phase of LiCoO (HT-LCO). Nanoscale HT-LCO is of interest to improve the battery performance as the lithium (Li) ion pathway is expected to be shorter in nanoparticles as compared to micron sized ones. Since batteries typically get recycled, the exposure to nanoparticles during this process needs to be evaluated.

Characteristics and properties of nano-LiCoO synthesized by pre-organized single source precursors: Li-ion diffusivity, electrochemistry and biological assessment.

Wear particles from automotive friction brake pads of various sizes, morphology, and chemical composition are significant contributors towards particulate matter. Knowledge concerning the potential adverse effects following inhalation exposure to brake wear debris is limited. Our aim was, therefore, to generate brake wear particles released from commercial low-metallic and non-asbestos organic automotive brake pads used in mid-size passenger cars by a full-scale brake dynamometer with an environmental chamber simulating urban driving and to deduce their potential hazard in vitro. The collected fractions were analysed using scanning electron microscopy via energy-dispersive X-ray spectroscopy (SEM-EDS) and Raman microspectroscopy. The biological impact of the samples was investigated using a human 3D multicellular model consisting of human epithelial cells (A549) and human primary immune cells (macrophages and dendritic cells) mimicking the human epithelial tissue barrier. The viability, morphology, oxidative stress, and (pro-)inflammatory response of the cells were assessed following 24 h exposure to ~ 12, ~ 24, and ~ 48 µg/cm of non-airborne samples and to ~ 3.7 µg/cm of different brake wear size fractions (2-4, 1-2, and 0.25-1 µm) applying a pseudo-air-liquid interface approach. Brake wear debris with low-metallic formula does not induce any adverse biological effects to the in vitro lung multicellular model. Brake wear particles from non-asbestos organic formulated pads, however, induced increased (pro-)inflammatory mediator release from the same in vitro system. The latter finding can be attributed to the different particle compositions, specifically the presence of anatase.

Biological response of an in vitro human 3D lung cell model exposed to brake wear debris varies based on brake pad formulation.

Inhalation of combustion-derived ultrafine particles (≤0.1 m) has been found to be associated with pulmonary and cardiovascular diseases. However, correlation of the physicochemical properties of carbon-based particles such as surface charge and agglomeration state with adverse health effects has not yet been established, mainly due to limitations related to the detection of carbon particles in biological environments. The authors have therefore applied model particles as mimics of simplified particles derived from incomplete combustion, namely, carbon nanodots (CNDs) with different surface modifications and fluorescent properties. Their possible adverse cellular effects and their biodistribution pattern were assessed in a three-dimensional (3D) lung epithelial tissue model. Three different CNDs, namely, nitrogen, sulfur codoped CNDs ( ) and nitrogen doped CNDs (  and ), were prepared by microwave-assisted hydrothermal carbonization using different precursors or different microwave systems. These CNDs were found to possess different chemical and photophysical properties. The surfaces of nanodots  and  were positively charged or neutral, respectively, arguably due to the presence of amine and amide groups, while the surfaces of  were negatively charged, as they bear carboxylic groups in addition to amine and amide groups. Photophysical measurements showed that these three types of CNDs displayed strong photon absorption in the UV range. Both  and  showed weak fluorescence emission, whereas  showed intense emission. A 3D human lung model composed of alveolar epithelial cells (A549 cell line) and two primary immune cells, i.e., macrophages and dendritic cells, was exposed to CNDs via a pseudo-air-liquid interface at a concentration of 100 g/ml. Exposure to these particles for 24 h induced no harmful effect on the cells as assessed by cytotoxicity, cell layer integrity, cell morphology, oxidative stress, and proinflammatory cytokines release. The distribution of the CNDs in the lung model was estimated by measuring the fluorescence intensity in three different fractions, e.g., apical, intracellular, and basal, after 1, 4, and 24 h of incubation, whereby reliable results were only obtained for . It was shown that  translocate rapidly, i.e., >40% in the basal fraction within 1 h and almost 100% after 4 h, while ca. 80% of the  and  were still located on the apical surface of the lung cells after 1 h. This could be attributed to the agglomeration behavior of  or . The surface properties of the  bearing amino and amide groups likely induce greater uptake as  could be detected intracellularly. This was less evident for , which bear carboxylic acid groups on their surface. In conclusion, CNDs have been designed as model systems for carbon-based particles; however, their small size and agglomeration behavior made their quantification by fluorescence measurement challenging. Nevertheless, it was demonstrated that the surface properties and agglomeration affected the biodistribution of the particles at the lung epithelial barrier .

Carbon nanodots: Opportunities and limitations to study their biodistribution at the human lung epithelial tissue barrier.

The overt hazard of carbon nanotubes (CNTs) is often assessed using in vitro methods, but determining a dose-response relationship is still a challenge due to the analytical difficulty of quantifying the dose delivered to cells. An approach to accurately quantify CNT doses for submerged in vitro adherent cell culture systems using UV-VIS-near-infrared (NIR) spectroscopy is provided here. Two types of multi-walled CNTs (MWCNTs), Mitsui-7 and Nanocyl, which are dispersed in protein rich cell culture media, are studied as tested materials. Post 48 h of CNT incubation, the cellular fractions are subjected to microwave-assisted acid digestion/oxidation treatment, which eliminates biological matrix interference and improves CNT colloidal stability. The retrieved oxidized CNTs are analyzed and quantified using UV-VIS-NIR spectroscopy. In vitro imaging and quantification data in the presence of human lung epithelial cells (A549) confirm that up to 85% of Mitsui-7 and 48% for Nanocyl sediment interact (either through internalization or adherence) with cells during the 48 h of incubation. This finding is further confirmed using a sedimentation approach to estimate the delivered dose by measuring the depletion profile of the CNTs.

Quantification of Carbon Nanotube Doses in Adherent Cell Culture Assays Using UV-VIS-NIR Spectroscopy.

: Pulmonary drug delivery is a complex field of research combining physics which drive aerosol transport and deposition and biology which underpins efficacy and toxicity of inhaled drugs. A myriad of preclinical methods, ranging from  to  and , can be implemented.: The present review covers  mathematical and computational fluid dynamics modelization of aerosol deposition, cascade impactor technology to estimated drug delivery and deposition, advanced  cell culture methods and associated aerosol exposure, lung-on-chip technology,  modeling,  inhaled drug delivery, lung imaging, and longitudinal pharmacokinetic analysis.: No single preclinical model can be advocated; all methods are fundamentally complementary and should be implemented based on benefits and drawbacks to answer specific scientific questions. The overall best scientific strategy depends, among others, on the product under investigations, inhalation device design, disease of interest, clinical patient population, previous knowledge. Preclinical testing is not to be separated from clinical evaluation, as small proof-of-concept clinical studies or conversely large-scale clinical big data may inform preclinical testing. The extend of expertise required for such translational research is unlikely to be found in one single laboratory calling for the setup of multinational large-scale research consortiums.

Innovative preclinical models for pulmonary drug delivery research.

Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1-30 μg/cm) using an air-liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte-derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs.

Article	Year
Combined exposure of diesel exhaust particles and respirable Soufrière Hills volcanic ash causes a (pro-)inflammatory response in an in vitro multicellular epithelial tissue barrier model. Particle and fibre toxicology\| PubMed ID: 27955700	2016
Human Asthmatic Bronchial Cells Are More Susceptible to Subchronic Repeated Exposures of Aerosolized Carbon Nanotubes At Occupationally Relevant Doses Than Healthy Cells. ACS nano\| PubMed ID: 28505409	2017
Characteristics and properties of nano-LiCoO synthesized by pre-organized single source precursors: Li-ion diffusivity, electrochemistry and biological assessment. Journal of nanobiotechnology\| PubMed ID: 28830490	2017
Biological response of an in vitro human 3D lung cell model exposed to brake wear debris varies based on brake pad formulation. Archives of toxicology\| PubMed ID: 29748788	2018
Carbon nanodots: Opportunities and limitations to study their biodistribution at the human lung epithelial tissue barrier. Biointerphases\| PubMed ID: 30205690	2018
Quantification of Carbon Nanotube Doses in Adherent Cell Culture Assays Using UV-VIS-NIR Spectroscopy. Nanomaterials (Basel, Switzerland)\| PubMed ID: 31835823	2019
Innovative preclinical models for pulmonary drug delivery research. Expert opinion on drug delivery\| PubMed ID: 32057260	2020
Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS nano\| PubMed ID: 32167743	2020

Hana Barosova

Biography

JoVE Journal Publications

Other Publications