Air-Liquid Interface: Relevant In Vitro Models for Investigating Air Pollutant-Induced Pulmonary Toxicity

Air-Liquid Interface: Relevant In Vitro Models for Investigating Air Pollutant-Induced Pulmonary... Abstract Air pollution leads to inhalation of several pulmonary stimulants that includes particulate matter, and gaseous substances contributing significantly to the development of chronic lung diseases. However, the pathophysiological mechanism of air pollutant mediated pulmonary toxicity remains unclear. This is primarily due to the lack of efficient test systems, mimicing human inhalation exposure scenarios to air pollutants. The majority of the pulmonary in vitro studies have been conducted using cell lines in submerged cell culture conditions and thereby overlooking the pulmonary physiology. Moreover, submerged cell culture systems lack the possibility to measure effective dose measurements. Particle properties, such as size, surface charge, solubility, transformation, or agglomeration state and chemical properties are altered in solution and are dependent on the composition of cell culture medium. Physiologically relevant in vivo-like in vitro models cultured at air-liquid interface (ALI) is therefore becoming a realistic and efficient tool for lung toxicity testing and cell-cell interaction studies following exposure to aerosolized or gaseous form of air pollutants. Primary bronchial epithelial cells cultured at ALI leads to differentiate into respiratory epithelium consisting of ciliated cells, goblet cells, club cells and basal cells. ALI system is also considered as a feasible approach to implement the “3R principle”—replacement, reduction, and refinement of animal usage in lung toxicity studies. This review discusses the current understanding of relevance, benefits and limitations of the ALI models in comparison to the existing in vitro and in vivo exposure system for testing air pollutants mediated pulmonary toxicity. air-liquid interface, air pollution, pulmonary toxicity Series of epidemiological studies reported that inhalation exposure to air pollutants (particles: particulate matter [PM] and gases) causes pulmonary toxicity and onset of chronic lung diseases like chronic obstructive pulmonary disease (COPD), chronic bronchitis and asthma (Esposito et al., 2014, Hoek et al., 2013). Respirable PM have been identified as a “criteria air pollutant” along with carbon monoxide, ground level ozone, nitrogen dioxide, sulfur dioxide and lead by the U.S.-Environmental Protection Agency (Lenz et al., 2013, Schmid and Stoeger, 2016, Teeguarden et al., 2007). Moreover, exponential growth in the use of nanomaterials during last decades presents the increased risk of human exposure to nanoparticles (NPs). This further escalated the NPs exposure mediated concern on public health and safety (Johnston et al., 2013). The surface area of human lung (80m2) is roughly same as the size of a tennis court (Notter, 2000). This represents lung as a major port of entry for small-sized particles and other gaseous pollutants to the human body (Ghio et al., 2012). Ultrafine or NP, by virtue of their small size and capacity to evade substantially the muco-ciliary and macrophagic clearance, can easily penetrate and get deposited in the alveolar region of the lungs (Latvala et al., 2016, Müller et al., 2010, Sorokin, 1970, Willers et al., 2013). Similar to PMs, exposure to toxic gaseous substances also remains an ongoing health concern for the assessment of environmental, occupational (industrial chemicals, cosmetics), intentional (tobacco smoke), and accidental exposure (Fröhlich and Meindl, 2015, Yaghi and Dolovich, 2016). Several researchers have established in vitro lung model of all the segments of respiratory system starting from nasal cavity, trachea, bronchial mucosa and down through airways to the alveolar region. Established in vitro models are being routinely used to study the air pollution mediated respiratory toxicity and lung diseases to improve our knowledge on molecular and patho-physiological mechanisms. The in vitro lung models range from simple cell-free biochemical assays (Mudway et al., 2004, Zielinski et al., 1999) to submerged mono-culture systems (Bhattacharya et al., 2013; Gliga et al., 2014; Nymark et al., 2013) to the most recent and advanced multi-cell-type cultures grown at air-liquid interface (ALI, Lenz et al., 2013, Müller et al., 2010). The advanced bronchial ALI models represent a fully differentiated epithelium with more than 1 cell type (ciliated cells, goblet cells, and basal cells) that are often combined with immunocompetent cells (dendritic cells, macrophages, neutrophiles). The potential toxicity of PM and gaseous pollutants has been widely tested in animal models and cell cultures systems (Fröhlich and Salar-Behzadi, 2014; Landsiedel et al., 2014; Sager et al., 2008) (Figure 1). Long-term inhalation exposure studies using rodents have been used to assess the air pollutants mediated human health risk. However, species specific differences always pose a concern while using animal models (Landsiedel et al., 2014). Anatomical, physiological, and molecular differences between humans and rodents does impact on particle deposition, transportation, and clearance mechanisms thereby often resulting in inaccurate pulmonary as well as systemic risk assessment (Clippinger et al., 2016; Latvala et al., 2016). Apart from that, animal experiments are of ethical concerns, expensive, time consuming and regulated by stricter laws than before (Figure 1). Figure 1. View largeDownload slide Strategic consideration to implement physiologically relevant in vitro risk assessment. COPD, Chronic obstructive pulmonary disease; ALI, air-liquid interface. Figure 1. View largeDownload slide Strategic consideration to implement physiologically relevant in vitro risk assessment. COPD, Chronic obstructive pulmonary disease; ALI, air-liquid interface. In contrast, in vitro systems are cost-effective, time efficient and easier to perform. The major advantages of in vitro research are the better scope to study detailed cellular and subcellular functions, cell-cell communication, and molecular mechanisms compared with animal models. Therefore, during the last few years significant efforts have been made to reduce, refine, and replace animal experiments by physiologically relevant respiratory mucosa models (Lee et al., 2009; Rothen-Rutishauser et al., 2008; Thai et al., 2005). However, the relevance of using single cell in vitro systems for performing cytotoxicity assessment of the respiratory system is questionable (Clippinger et al., 2016; Fröhlich and Salar-Behzadi, 2014). Thus, a careful scrutinization of the relevance of currently used in vitro models for predicting pulmonary toxicity is warranted. In this review, we evaluated the current practices to assess NP and gaseous substance mediated in vitro pulmonary toxicity testing. We have discussed the advantages and limitations of the existing submerged and advanced ALI models developed with primary lung cells or relevant cell lines in terms of dosimetry and physiological relevance. Additionally, we have compared the commercially available and laboratory developed exposure systems with in-built dosimetry measurement for NPs or gases. SUBMERGED CELL CULTURE Lack of Physiological Relevance Submerged in vitro models lack the physiological features of airway mucosa. The airway consists of more than 40 different cell types and the thickness of epithelial cell layer also varies at different airway regions along with the presence of different cell types (Sorokin, 1970; Stoeger et al., 2005). The bronchial surface under in vivo condition is lined by a pseudostratified epithelium, characterized by ciliated columnar cells interspersed with mucous producing goblet cells higher up in the airways and further down cuboidal ciliated cells are present with the club cells with the capacity to produce club cell protein (Müller et al., 2010). Further down the alveolar epithelial cell layer is very thin consisting of alveolar type I cells and surfactant producing alveolar type II cells in close connection with endothelial cells to facilitate the oxygen exchange (Fahy and Dickey, 2010). Immunocompetent cells like resident macrophages, dendritic cells and neutrophils are also present. Presence of the surfactant film over throughout the airways epithelial surface plays a critical role in particle movement and retention properties. Therefore, lung cells cultured under submerged conditions do not mimic in vivo condition. Dosimetry Most of the in vitro studies to assess pulmonary cytotoxicity have been performed using lung cells (cell lines and primary cells) cultured under submerged conditions (Akhtar et al. 2014). Stimulants (PMs/NPs, gases) are typically added directly into the cell culture media. However, such experimental set-ups do not reflect realistic cell-cell communication and cell-particle interactions similar to the real-life scenarios (Joris et al., 2013; Paur et al., 2011). As for example, addition of NPs directly into the cell culture medium increases the possibility of particle agglomeration, resulting in the exposure of cells to larger particles with smaller BET (Brunauer-Emmett-Teller) surface area, which is directly connected to particle toxicity (Lenz et al., 2013; Limbach et al., 2005). Particle BET surface area is considered to be one of the most important reference parameter for the characterization of detrimental toxic effects (inflammation and oxidative stress) caused by insoluble NPs (Schmid and Stoeger, 2016). Similar limitations have also been noted in case of toxicity assessment of gaseous pollutants in submerged cell culture, where stimulants are directly added directly to the cell culture medium. It has been well established that the properties of single NPs (movement or agglomeration status) are altered in the presence of cell culture medium (Limbach et al., 2005; Teeguarden et al., 2007) due to interaction with culture medium components, which may form a medium-specific corona (Loret et al., 2016; Lundqvist et al., 2011; Monopoli et al., 2011). The pathological and functional responses of lung cells to particles are also highly dependent on the physicochemical properties of NPs in association with direct interactions of particles on the state and activity of each individual cell (Rothen-Rutishauser et al., 2008). Therefore, any changes of particle properties after addition in the cell culture medium can highly influences the NPs-induced cytotoxicity (Lenz et al., 2013; Monopoli et al., 2011; Stoeger et al., 2006). All the above-mentioned parameters of NPs may be altered in suspended form when used under submerged cell culture-conditions. This may lead to unreliable outcomes and significantly limit the reproducibility of the system for further investigation (Lee et al., 2009). One of the main limitation of cells cultured at submerged conditions is to assess of the exact particle dose available for interaction with the cells (Lenz et al., 2013). This is due to the lack of direct methods for calculating particle number from the hydrodynamic particle properties, like size, density and shape. Moreover, a substantial fraction of the particles remains in the liquid or are lost in the lateral walls of the culture vessel, thereby influencing the actual dose of particles as well as their interaction with the cells. AIR-LIQUID INTERFACE Exposure experiments to air pollutants (PM/NPs and gases) under submerged cell culture conditions do not accurately address cell-cell and cell-stimulant interaction apart from dosimetry-related issues (Ahmad et al., 2014; Clippinger et al., 2016; Gminski et al., 2010; Lenz et al., 2013). To overcome the drawback related to physiological relevance of human airway mucosa, more complex models including multiple cells cultured at ALI (Lee et al., 2009; Rothen-Rutishauser et al., 2008; Thai et al., 2005) combined with advanced exposure systems to ensure accurate dosimetry (Ji et al., 2017; Klein et al., 2013; Rothen-Rutishauser et al., 2008) have been developed. In vitro exposure systems that deliver aerosolized pollutants to the surface of the cells cultured at ALI are of particular importance (Paur et al., 2011). In most of the exposure systems developed till date, deposition of aerosolized particles on cells occurs through the processes of diffusion and/or gravitation. Due to technical limitations, the maximum achieved dose using these systems remain generally far too low compared with the corresponding ambient pollutant concentration. Several factors influence the successful development of ALI-models that include choice of cell line, source of primary cells, coculture systems, mimicking preexisting conditions like chronic bronchits, dosimetry and customized cell culture conditions (medium, supplements, labware [Clippinger et al., 2016; Rothen-Rutishauser et al., 2008]). ALI system is also considered as a feasible approach to implement the “3 R principle”—replacement, reduction, and refinement of animal usage in pulmonary toxicity studies. Cell Line-Based ALI Model Different cell types like immortalized respiratory cell lines (eg, BEAS-2B and 16HBE140o, Calu-3, A549, NCI-H441) have been used to develop ALI models representing the human pulmonary system (Clippinger et al., 2016; Haghi et al., 2014; Zscheppang et al., 2018). Cell lines are homogenous and offer several advantages such as easy accessibility and can be use at high passages. However, respiratory cell lines on the other hand also exhibit some distinct limitations, such as their ability to produce mucus and form tight junctions. Similarly, quality assurances of the cell lines are also dependent on the source organization. American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures are popular among researchers because of their quality control by standardized protocol (eg, storage) for the development and maintenance of the cell lines (Clippinger et al., 2016). The Calu-3 cell line is of human lung adenocarcinoma; derived from metastatic site origin, commercially available from the ATCC and displays epithelial morphology as well as adherent growth (Forbes, 2000; Rothen-Rutishauser et al., 2008). The presence of tight junctions and the secretory activity makes the Calu-3 cell line a promising tool for lung drug absorption studies. BEAS-2B cell line was first developed from normal human epithelium and have been used to study airway structure and function on regular basis, however this cells line does not form tight junction (Clippinger et al., 2016; Zscheppang et al., 2018). Similarly, 16HBE is also a normal human airway epithelial cell line, which form extensive tight junction, but having less mucus producing ability compare to primary bronchial epithelial cells (PBECs) (Rothen-Rutishauser et al., 2008; Zscheppang et al., 2018). To develop alveolar airway mucosa A549 and NCI-H441 cell lines are mostly used. A549 cell lines are derived from pulmonary adenocarcinoma, which is consequently characterized as being representative of the alveolar type II pneumocytes of the human lung and are one of the most commonly used cell line in toxicity studies (Braakhuis et al., 2015; Rothen-Rutishauser et al., 2008; Zscheppang et al., 2018). Although, A549 cells produce surfactant protein, but they are not able to develop tight junction (Rothen-Rutishauser et al., 2008; Elbert et al. 1999). Whereas, for ion transport studies NCI-H441 cells are better choice since those cells form polarized tight monolayers and express organic cation transporters with P-glycoprotein (Rothen-Rutishauser et al., 2008; Zscheppang et al., 2018). Many of the widely used cell lines (Calu-3, A549) are carcinogenic in origin and likely to have altered genetic and phenotypic characteristics compared with normal cells. Therefore, this has led to a more conservative view on the cell line’s for suitability to recapitulate the bronchial and/alveolar epithelial cells phenotype. Primary Cell-Based ALI Model The use of primary human/animal cells cultured at ALI is considered as a better condition than using cell lines. The toxicological findings on the basis of primary human cells at ALI condition are more acceptable to predict the regulatory assessment compared with primary animal cells (Gruber and Hartung 2004; Rothen-Rutishauser et al., 2008). The cells isolated from fresh animal tissue (primary culture) represent a more heterogeneous population of different cell types, hence isolation and identification of specific cell types need to be performed carefully during establishment of methodology (Zscheppang et al., 2018). Use of primary human lung cells is limited due to the availability of human pulmonary tissue and their (donor) variation. However, human bronchial epithelial cells from normal and diseased donors as well as human lung microvascular endothelial cells are available from commercial sources. Thai and co-workers (Kao et al., 2005; Thai et al., 2005) have developed ALI-models using both human and mice primary airway epithelial cells. The establishment of ALI-models is highly dependent on the use of animal-derived media supplements, such as bovine pituitary extract and growth factors. Another crucial factor for the development of physiologically relevant in vitro model is the differentiation of epithelium consisting of basal cells, ciliated cells, and goblet cells that are present in vivo (Zscheppang et al., 2018). To overcome the limitation of availability of primary cells, many researchers use the commercially available ALI-models. The human airway models (EpiAirway) developed by MatTek’s (MatTek Corporation, MA, USA), OncoCilAirTM and MucilAirTM (Epithelix Sarl, Suisse) are having more than 30 donors from healthy and diseased individuals (asthmatics, COPD patients, and smoker). These commercially available models can be used for toxicity screening, risk assessment, preliminary drug discovery studies and also the exploration of molecular pathways of specific disease (Zscheppang et al., 2018). The main advantage of the commercially available ALI-models is their long lifetime (3–12 months), allowing the possibility for repeated or chronic exposure studies. However, the commercially available systems are limited in terms of flexibility (cell manipulation, genetic manipulation) and customization (disease-specific donor for primary cells (Zscheppan et al., 2018). Coculture System Introduction of multicellular ALI-models are considered an advancement of cell-cell communications comparable with in vivo condition (Fröhlich and Salar-Behzadi 2014). Therefore, the in vitro multi-cellular airway mucosa ALI-models that include different cell types (epithelial cells, macrophages, endothelial cells, and/or dendritic cells) are considered to be with high physiological relevance (Klein et al., 2013). Exposure of ALI-models to aerosolized PM or gaseous pollutants allows the direct interaction of particles and gases with cells surface representing in vivo situation (Ahmad et al., 2014; Gostner et al., 2016; Holder et al. 2008; Jing et al., 2015). In this aspect, another important physiological phenomenon is the formation of the thin liquid lining layer with surfactant mucus and the ciliary movement that is important for mucociliary clearance. Both surfactant and mucociliary clearance plays crucial role in particle displacement and retention (Möller et al., 2008; Rothen-Rutishauser et al., 2008). Another advanced multicellular ALI-model including A549 human blood monocyte-derived macrophages and dendritic cells has been developed by Rothen-Rutishauser et al. (2005), The A549 cells required to be cultured for adequate time at ALI to be able to produce surfactant. An alveolar ALI system consisting of 4 different human cell lines representing cell-cell response of the alveolar surface have been developed by Klein et al. (2013) to study the effect of aerosolized NPs. In this system endothelial cells (EA.hy 926) were seeded on the basolateral side of a micro porous membrane. Epithelial cells (A549) together with innate immune cells (mast cells: HMC-1 and macrophage-like cells: THP-1) were seeded on the apical compartment of the transwell inserts. These cocultures were designed in order to develop the 3D-organization of the cells, resembling the structure of alveolar barrier. Comparison of inflammatory and oxidative stress response between A549 cell lines and primary human PBECs cultured at ALI following exposure to CuO NPs under identical exposure concentration was performed. A549 cells were found to be more susceptible to CuO NP-mediated toxicity compared with PBEC (Jing et al., 2015). However, in order to mimic more closely the in vivo situations and to maximize the human relevance of in vitro testing, the combinations of complex cellular in vitro models with precise dosimetry calculations following exposure to aerosolized materials are in urgent need (Holder et al. 2008; Loret et al., 2016). Diseased ALI Model Ji et al. (2017) have recently shown the successful development of both normal and chronic bronchitis-like ALI-models using human PBEC. Treatment of PBEC with interleukin 13 (IL-13) at ALI condition resulting chronic bronchitis-like mucosa due to the generation of more mucus producing cells, a characteristic feature of chronic bronchitis. The study compared the effects of NPs exposure on both normal and chronic bronchitis-like diseased airway mucosa. These physiologically relevant ALI-models in combination with an exposure system that allows delivery of dry aerosolized NPs similar to inhalation exposure of humans (Ji et al., 2017; Zanoni et al., 2016). Similarly, several research groups have developed tumor spheroid models to study tumor growth, drug testing, in which a cluster of cells are grown together to form a viable tumour-like 3D structure (Zanoni et al., 2016; Zscheppang et al., 2018,). Thai et al. (2005) have developed ALI-models using both human and mouse primary epithelial cells to study the effect of IL-13 on 2 biomarkers, MUC5AC and hCLCA1/Gob5, which are frequently associated with surface mucous and goblet cells in asthmatic airways. However, development of ALI-model is time consuming and the availability of the primary cells from human donors is limited. This is coupled with a high variation of interindividual response to stimulant (Figure 2). Figure 2. View largeDownload slide Advancement of in vitro exposure model from submerged monoculture to physiologically relevant ALI model in association with exposure technique or exposure system to different environmental stimulants. Figure 2. View largeDownload slide Advancement of in vitro exposure model from submerged monoculture to physiologically relevant ALI model in association with exposure technique or exposure system to different environmental stimulants. Direct Comparisons of Particle Exposure in Submerged and ALI Culture Conditions Lenz et al. (2013) have investigated the cellular response of human alveolar epithelial-like cells (A549) cultured simultaneously at ALI and under submerged condition after exposure to airborne zinc oxide (ZnO) NPs. The findings of the study showed that cells exposed to ZnO-NPs at ALI system depicts significantly consistent cellular response in terms of oxidative stress and inflammation compared with submerged cultures. This observation is comparable with other literature data suggested that in vitro toxicity screening of NP with ALI-models may produce less false negative results than screening with submerged cell cultures (Lenz et al., 2013; Wilkinson et al., 2011). Culturing of PBECs at ALI followed by exposure to palladium nanoparticles (PdNPs) showed low uptake (Ji et al., 2017) in contrast to exposing the PBECs to PdNPs under submerged condition, where particles uptake was observed within 1 h after addition to the cultures (Wilkinson et al., 2011). The lack of particles in ALI-models could be explained by an existing mucociliary clearance. Direct Comparisons of Gaseous Air Pollutant Exposure in Submerged and ALI Culture Conditions Chronic or long-term exposure to gaseous air pollutants may be responsible for the onset of long-term respiratory effects like asthma, allergy and even onset of neurological disorders (Salthammer and Bahadir, 2009). Formaldehydes, carbon monoxide and ozone are the most commonly detected compounds in indoor environment and are also responsible for the development of acute toxicity (respiratory irritation; Petry et al., 2004; Salthammer and Bahadir, 2009). However, the major challenge in traditional submerged in vitro model is the available exposure system to expose the cells to gaseous or vaporized stimulants (Tsoutsoulopoulos et al., 2016). In most of the studies, toxicity assessment of gases is performed by direct addition of test compound (in liquid form) to the cell culture medium. Similar to the particles, the addition of chemicals in the medium may alter the properties of the stimulant due to interactions and binding with various components of the medium thereby resulting in unreliable outcomes (Gminski et al., 2010). Therefore, to test the cellular effect of gases in the gaseous phase, advanced in vitro ALI-model in combination with exposure systems capable of gaseous exposure are recommended (Dwivedi et al., 2018). However, there are only few exposure systems that are commercially available to suit the purpose allowing exposure of cultured cells to gas with a precise dosimetry and without any interfering medium (Gostner et al., 2016). The study by Ahmad et al. (2014) compared the variabilities in toxicity assessment of chlorine by using submerged and ALI-models. They demonstrated that chlorine reacts rapidly with aqueous surfaces to form hydrochloric and hypochlorous acids; and therefore, under submerged condition the observed toxicity effect might only demonstrated the effects of by-products such as hydrochloric acid rather than chlorine itself. Whereas, exposure of differentiated ALI cultures of human airway epithelial cells to chlorine gas allows a direct interaction of chlorine with cell surface in the absence of aqueous media. This is comparable to realistic exposure scenarios through inhalation of different gases in environmental and occupational settings. In a recent study (Dwivedi et al., 2018), ALI-models (including differentiated human PBEC together with fibroblasts) combined with a newly developed exposure system were exposed to gaseous form of acrolein, crotonaldehyde and hexanal. In this study we detected significant induction of inflammatory response was detected in association with elevated oxidative stress following exposure of human PBEC cultured at ALI to 3 different aldehydes (acrolein, crotonaldehyde, and hexanal) in gaseous form. Addition of these aldehydes directly to the cell culture medium resulted in nonsignificant alteration of inflammatory or oxidative stress response. The inflammatory response observed in ALI-models corresponded well with previous in vivo studies. This indicates that lung cells cultured at ALI are more suitable than the submerged in vitro model for toxicity assessment studies following exposure to aldehydes. The exposure chambers used in this study for exposing PBEC cultured at ALI to the aldehydes in gaseous form has been developed in our laboratory (Dwivedi et al., 2018). The steady target concentration of aldehydes was achieved by injection of an appropriate amount of aldehyde in the tedlar bag and the aldehyde was sucked in with clean air at a high flow rate into the exposure chamber. The actual concentration of aldehyde was monitored in real time using an attached gas chromatography just prior to each exposure (Dwivedi et al., 2018). The toxicological evaluation of tobacco whole-smoke (TWS) has been carried out with the primary focus on particulate fraction, which is often collected by passing TWS through a filter pad (Weber et al., 2013). The particulate fraction constitutes only 5%–10% of the total TWS generated from burning tobacco (Keith and Tesh, 1965). Hence the remaining compounds of TWS remain unassessed for toxicity evaluation. With the advancement of technology, such problems have been significantly resolved by preparing aqueous extracts of TWS by bubbling through a biological buffer or cell culture medium. However, due to the complexity and chemical composition, many compounds present in TWS remains undissolved in the biological buffer or in cell culture medium (Azzopardi et al., 2015; Thorne and Adamson, 2013). Therefore, in order to completely assess toxicological effects of TWS in the lung, an ideal in vitro cell culture system in association with an exposure system capable of delivering TWS in gaseous form including the particle fraction to lung cells exposed at ALI condition is required. Schamberger et al. (2015) has shown that chronic exposure of PBEC at ALI to cigarette smoke extract (CSE) through basal media significantly impair the cellular composition of the airway epithelium with reduced number of ciliated cells and increased number of club and goblet cells. Moreover, chronic exposure of PBEC to CSE extract has been associated with reduced cilia length and reduction in total acetylated tubulin (Schamberger et al., 2015). ALI-models used in this study are physiologically the most relevant in vitro models, and provides a suitable tool to study in detail the cellular effect observed in individuals with chronic lung diseases. However, exposure of these models to cigarette smoke instead of CSE might have been more appropriate to compare with the observed effects in vivo. Extensive developments of both in vitro ALI-models and the exposure system to deliver tobacco smoke have been observed during the last few years. The combination of these 2 criteria may finally allow to develop a well-defined uniform deposition system for TWS aerosol on pulmonary cells cultured in physiologically relevant in vitro models. Azzopardi et al. (2015) have shown such a combination by using Borgwaldt RM20S smoking machine combined with bronchial epithelial cell line (NCI-H292) cultured at ALI. In this study the investigators have demonstrated the sensitivity of epithelial cells line (NCI-H292) cultured at ALI by measuring cytotoxicity and inflammatory response following exposure to TWS and tobacco vapor (Azzopardi et al., 2015). The authors have shown the advantages of ALI-models in combination with this advanced exposure system in order to assess the sensitivity response of TWS derived from 2 different smoking regiments (Health Canada Intense and International Organization for Standard). AVAILABLE AIR POLLUTANT (PARTICLES AND GAS) EXPOSURE SYSTEM FOR ALI MODELS Many methods have been used to expose cells cultured at ALI to aerosolized stimulant either in dry powder form or by nebulization of solutions. The most crucial part of these exposure systems is to ensure the homogenous and even distribution of particles over the whole cell surface and reduced agglomeration of particles to avoid their alteration of physicochemical properties. Many research groups assessed the effects of air pollutants using diffusion exposure chambers. However, exposure of ALI-models to aerosolized particles or gases need to be further developed with easy and precise dosimetry system. The exposure systems developed specifically for ALI-based cell culture systems have been used to test several toxic substances such as different gases, copper NPs, carbon NPs, ZNO NPs, gold NPs, polystyrene NPs, cerium oxide NPs, and laser printer emission particles (Fuchs, 2002; Imanishi et al., 2009; Lenz et al., 2009). But standardized quantification of the deposited aerosols is essential as aerosols or NPs contained in the aerosol can be retained by components of the exposure systems or may form agglomeration which may result in undesired interaction and causing limitations of the outcomes. The most commonly used commercially developed in vitro particle exposure systems are from VITROCELL and CULTEX. In both these systems where the cells co-cultured at ALI and aerosolized particles or gas exposure takes place for 15–60 min under humid atmosphere at a temperature of 37°C (Gervelas et al., 2007; Niwa et al., 2007). Particle deposition is mostly driven by sedimentation and/or diffusion. Other established and commercially available in vitro aerosol exposure systems like CULTEX (Cultex Laboratories GmbH, Hannover, Germany) and electrostatic aerosol in vitro exposure system uses electrostatic precipitation of aerosol over the cells cultured at ALI condition (Aufderheide and Mohr, 2000; Niwa et al., 2007; Ritter et al., 2003). Exposure of ALI model to aerosolized air pollutants by these devices are technically easy to perform, but the deposition rates in these devices exhibit considerable variation. The obtained variation in deposition rate is probably related to the aerosolization technique (Sorokin 1970). Among the above described exposure systems, the major challenge however are concerning the formation of particle agglomeration with long exposure duration. Loading large amount of exposure materials (particles/gases) may also pose a technical hurdle, while minimal amount (1 mg) of particle loading is linked to the accuracy of the analytical balance and to achieve the target dose with a specific time duration (Brandenberger et al., 2010; Sorokin, 1970). MicroSprayer IA-1 C aerosolizer, a very small number of NPs are required to reach the target dose and the system shows less electrostatic interaction between particles and devices thereby represent a low risk of particle aggregation. However, the major disadvantage of this exposure system is the formation of droplets which may affect the homogeneous deposition of particles on in vitro model (Sorokin, 1970). Other noncommercial exposure systems with a limited number of users have been also described. These include the Biological aerosol trigger, Voisin chamber, Minu cell system, Nano Aerosol Chamber In vitro Toxicity, air-liquid interface cell exposure (ALICE) system. However, these exposure systems need further modification to standardize the particle deposition and dosimetry. ALICE system is an aerosol exposure instruments that are specially developed for exposure to aerosolized NPs, and has also been used by several investigators (Ritter et al., 2003). In ALICE exposure system, a dense cloud of droplets is generated which are finally transported to the exposure chamber with a specific flow rate. The generated droplets deposit uniformly over the cell surface due to single particle sedimentation by a process called cloud settling. ALICE exposure system allows a uniform, efficient and dose-controlled deposition of NP suspensions onto the cell surfaces at ALI; however the particle loading in suspension form may have an effect on the physicochemical characteristics of particles as already discussed (Fuchs, 2002). In spite of this apparent limitation, this particular exposure set-up might be considered as an optimal system to screen toxicity of NPs used in pharmaceutical industries where suspension-based aerosolized NP are used in the drug formulations; whereas in environmental or occupational exposure scenarios dry aerosolized NP are a more realistic exposure route (Duret et al., 2012). Recently, Ji et al. (2017) used an advanced exposure system (XposeALI) for exposure of ALI-models to dry aerosolized NP (Ji et al., 2017). The XposeALI system offers the scope for exposing ALI-models to dry aerosolized environmental pollutants. In this exposure system, aerosolized NPs are mainly generated by pushing small batches of dry NP powder through a high-pressure aerosol generator into a 300-ml holding chamber of the exposure system. Finally, the generated aerosol is pumped from the holding chamber through a Casella light dispersion instrument and dispersed uniformly into the exposure manifold, at a main flow rate of 120 ml/min. The main flow is diverted into 3 consecutive branch flows for individual exposure of 3 cell culture inserts at the time of exposure (Ji et al., 2017). This particular commercially available system is suitable for exposure of ALI cell culture inserts to aerosolized NPs, and it is also possible to compare and validate the in vitro findings with in vivo inhalation (rodents) exposure by using the same instrument but with different exposure modules. However, most of these exposure systems exhibit considerable variations in deposition rates, which might be due to different aerosolization technique and may also relate to particle properties (eg, size, carbon NPs, or metal NPs, etc). Therefore, further development of all of the discussed exposure system is required to avoid variation in deposition rate with particle properties. FUTURE DEVELOPMENT OF IN VITRO MODEL AND EXPOSURE SYSTEM The enormous effort made by many investigators have resulted an extensive advancement of the in vitro models and its usage at different levels of research covering toxicity screening to detailed molecular signaling pathway studies (Figure 2). Due to the emergence of nanotechnology and release of NPs in the environment from different sources (like automobile exhaust and abrasion of car tyres), there is an urgent need to understand the details of the NP- human body interaction and the plausible molecular mechanism for health risk assessment. In vitro models and particularly ALI-models mimicking human airways are of valuable support for the toxicological assessments of air pollutants. Further development of these systems is required to investigate cellular and adaptive responses through long term or chronic exposures and combined exposure studies of both NPs and gases. Additionally, ALI models of different levels of the lung (bronchial vs alveolar mucosa) as well as other organs (ALI model of gut or skin) need to be developed for further risk assessment. In the current review we have highlighted the different airway mucosa models and the corresponding exposure systems available commercially and noncommercially. Each of those exposure systems are unique in their own way and may require standardization at the next level to deliver both NPs and gases even with more precise dosimetry for in vitro studies. In vitro model is an important platform to screen cellular pathogenesis of air pollutant-mediated lung diseases; however, in vitro system lacks the complexity of the entire organism. Therefore, investigation and validation of current in vitro findings with in vivo outcomes, which includes detailed studies of air pollutants (particles and gases) induced toxicity and cellular responses in animal models (eg, rodents) using same exposure system are warranted. However, most of the exposure systems mentioned in this review are mainly developed for in vitro exposure studies. Whereas, XposeALI, is one of the commercially available platform that offers both in vitro and in vivo exposure to dry aerosolized NPs under identical exposure conditions.The validation of in vitro findings compared with in vivo outcomes will significantly enhance the strength of the in vitro data. This will allow researchers to perform cross-platform comparisons for further progression of translational research and will be the most important feasible approach to implement 3R strategy of animal model to perform air pollutant mediated pulmonary toxicity studies. FUNDING Swedish Fund for Research without Animal Experiments (22/10, 40/11, F35/12, F25/13, F34-14, F36/15); Swedish Research Council (VR 521 2010-2801, 2014-02767); Swedish Heart-lung foundation (20100180, 20120376, 20120818, 20150328, 20150329, 20150330); European Respiratory Society (ERS: ERS LTRF 2014-3567); Karolinska Institutet, Sweden. REFERENCES Ahmad S. , Ahmad A. , Neeves K. B. , Hendry-Hofer T. , Loader J. E. , White C. W. , Veress L. ( 2014 ). In vitro cell culture model for toxic inhaled chemical testing . J. Vis. Exp . 87 , 51539 . Akhtar M. J. , Ahamed M. , Khan M. A. , Alrokayan S. A. , Ahmad I. , Kumar S. ( 2014 ). Cytotoxicity and apoptosis induction by nanoscale talc particles from two different geographical regions in human lung epithelial cells . Environ. Toxicol . 29 , 394 – 406 . Google Scholar CrossRef Search ADS PubMed Aufderheide M. , Mohr U. ( 2000 ). 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Air-Liquid Interface: Relevant In Vitro Models for Investigating Air Pollutant-Induced Pulmonary Toxicity

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Abstract

Abstract Air pollution leads to inhalation of several pulmonary stimulants that includes particulate matter, and gaseous substances contributing significantly to the development of chronic lung diseases. However, the pathophysiological mechanism of air pollutant mediated pulmonary toxicity remains unclear. This is primarily due to the lack of efficient test systems, mimicing human inhalation exposure scenarios to air pollutants. The majority of the pulmonary in vitro studies have been conducted using cell lines in submerged cell culture conditions and thereby overlooking the pulmonary physiology. Moreover, submerged cell culture systems lack the possibility to measure effective dose measurements. Particle properties, such as size, surface charge, solubility, transformation, or agglomeration state and chemical properties are altered in solution and are dependent on the composition of cell culture medium. Physiologically relevant in vivo-like in vitro models cultured at air-liquid interface (ALI) is therefore becoming a realistic and efficient tool for lung toxicity testing and cell-cell interaction studies following exposure to aerosolized or gaseous form of air pollutants. Primary bronchial epithelial cells cultured at ALI leads to differentiate into respiratory epithelium consisting of ciliated cells, goblet cells, club cells and basal cells. ALI system is also considered as a feasible approach to implement the “3R principle”—replacement, reduction, and refinement of animal usage in lung toxicity studies. This review discusses the current understanding of relevance, benefits and limitations of the ALI models in comparison to the existing in vitro and in vivo exposure system for testing air pollutants mediated pulmonary toxicity. air-liquid interface, air pollution, pulmonary toxicity Series of epidemiological studies reported that inhalation exposure to air pollutants (particles: particulate matter [PM] and gases) causes pulmonary toxicity and onset of chronic lung diseases like chronic obstructive pulmonary disease (COPD), chronic bronchitis and asthma (Esposito et al., 2014, Hoek et al., 2013). Respirable PM have been identified as a “criteria air pollutant” along with carbon monoxide, ground level ozone, nitrogen dioxide, sulfur dioxide and lead by the U.S.-Environmental Protection Agency (Lenz et al., 2013, Schmid and Stoeger, 2016, Teeguarden et al., 2007). Moreover, exponential growth in the use of nanomaterials during last decades presents the increased risk of human exposure to nanoparticles (NPs). This further escalated the NPs exposure mediated concern on public health and safety (Johnston et al., 2013). The surface area of human lung (80m2) is roughly same as the size of a tennis court (Notter, 2000). This represents lung as a major port of entry for small-sized particles and other gaseous pollutants to the human body (Ghio et al., 2012). Ultrafine or NP, by virtue of their small size and capacity to evade substantially the muco-ciliary and macrophagic clearance, can easily penetrate and get deposited in the alveolar region of the lungs (Latvala et al., 2016, Müller et al., 2010, Sorokin, 1970, Willers et al., 2013). Similar to PMs, exposure to toxic gaseous substances also remains an ongoing health concern for the assessment of environmental, occupational (industrial chemicals, cosmetics), intentional (tobacco smoke), and accidental exposure (Fröhlich and Meindl, 2015, Yaghi and Dolovich, 2016). Several researchers have established in vitro lung model of all the segments of respiratory system starting from nasal cavity, trachea, bronchial mucosa and down through airways to the alveolar region. Established in vitro models are being routinely used to study the air pollution mediated respiratory toxicity and lung diseases to improve our knowledge on molecular and patho-physiological mechanisms. The in vitro lung models range from simple cell-free biochemical assays (Mudway et al., 2004, Zielinski et al., 1999) to submerged mono-culture systems (Bhattacharya et al., 2013; Gliga et al., 2014; Nymark et al., 2013) to the most recent and advanced multi-cell-type cultures grown at air-liquid interface (ALI, Lenz et al., 2013, Müller et al., 2010). The advanced bronchial ALI models represent a fully differentiated epithelium with more than 1 cell type (ciliated cells, goblet cells, and basal cells) that are often combined with immunocompetent cells (dendritic cells, macrophages, neutrophiles). The potential toxicity of PM and gaseous pollutants has been widely tested in animal models and cell cultures systems (Fröhlich and Salar-Behzadi, 2014; Landsiedel et al., 2014; Sager et al., 2008) (Figure 1). Long-term inhalation exposure studies using rodents have been used to assess the air pollutants mediated human health risk. However, species specific differences always pose a concern while using animal models (Landsiedel et al., 2014). Anatomical, physiological, and molecular differences between humans and rodents does impact on particle deposition, transportation, and clearance mechanisms thereby often resulting in inaccurate pulmonary as well as systemic risk assessment (Clippinger et al., 2016; Latvala et al., 2016). Apart from that, animal experiments are of ethical concerns, expensive, time consuming and regulated by stricter laws than before (Figure 1). Figure 1. View largeDownload slide Strategic consideration to implement physiologically relevant in vitro risk assessment. COPD, Chronic obstructive pulmonary disease; ALI, air-liquid interface. Figure 1. View largeDownload slide Strategic consideration to implement physiologically relevant in vitro risk assessment. COPD, Chronic obstructive pulmonary disease; ALI, air-liquid interface. In contrast, in vitro systems are cost-effective, time efficient and easier to perform. The major advantages of in vitro research are the better scope to study detailed cellular and subcellular functions, cell-cell communication, and molecular mechanisms compared with animal models. Therefore, during the last few years significant efforts have been made to reduce, refine, and replace animal experiments by physiologically relevant respiratory mucosa models (Lee et al., 2009; Rothen-Rutishauser et al., 2008; Thai et al., 2005). However, the relevance of using single cell in vitro systems for performing cytotoxicity assessment of the respiratory system is questionable (Clippinger et al., 2016; Fröhlich and Salar-Behzadi, 2014). Thus, a careful scrutinization of the relevance of currently used in vitro models for predicting pulmonary toxicity is warranted. In this review, we evaluated the current practices to assess NP and gaseous substance mediated in vitro pulmonary toxicity testing. We have discussed the advantages and limitations of the existing submerged and advanced ALI models developed with primary lung cells or relevant cell lines in terms of dosimetry and physiological relevance. Additionally, we have compared the commercially available and laboratory developed exposure systems with in-built dosimetry measurement for NPs or gases. SUBMERGED CELL CULTURE Lack of Physiological Relevance Submerged in vitro models lack the physiological features of airway mucosa. The airway consists of more than 40 different cell types and the thickness of epithelial cell layer also varies at different airway regions along with the presence of different cell types (Sorokin, 1970; Stoeger et al., 2005). The bronchial surface under in vivo condition is lined by a pseudostratified epithelium, characterized by ciliated columnar cells interspersed with mucous producing goblet cells higher up in the airways and further down cuboidal ciliated cells are present with the club cells with the capacity to produce club cell protein (Müller et al., 2010). Further down the alveolar epithelial cell layer is very thin consisting of alveolar type I cells and surfactant producing alveolar type II cells in close connection with endothelial cells to facilitate the oxygen exchange (Fahy and Dickey, 2010). Immunocompetent cells like resident macrophages, dendritic cells and neutrophils are also present. Presence of the surfactant film over throughout the airways epithelial surface plays a critical role in particle movement and retention properties. Therefore, lung cells cultured under submerged conditions do not mimic in vivo condition. Dosimetry Most of the in vitro studies to assess pulmonary cytotoxicity have been performed using lung cells (cell lines and primary cells) cultured under submerged conditions (Akhtar et al. 2014). Stimulants (PMs/NPs, gases) are typically added directly into the cell culture media. However, such experimental set-ups do not reflect realistic cell-cell communication and cell-particle interactions similar to the real-life scenarios (Joris et al., 2013; Paur et al., 2011). As for example, addition of NPs directly into the cell culture medium increases the possibility of particle agglomeration, resulting in the exposure of cells to larger particles with smaller BET (Brunauer-Emmett-Teller) surface area, which is directly connected to particle toxicity (Lenz et al., 2013; Limbach et al., 2005). Particle BET surface area is considered to be one of the most important reference parameter for the characterization of detrimental toxic effects (inflammation and oxidative stress) caused by insoluble NPs (Schmid and Stoeger, 2016). Similar limitations have also been noted in case of toxicity assessment of gaseous pollutants in submerged cell culture, where stimulants are directly added directly to the cell culture medium. It has been well established that the properties of single NPs (movement or agglomeration status) are altered in the presence of cell culture medium (Limbach et al., 2005; Teeguarden et al., 2007) due to interaction with culture medium components, which may form a medium-specific corona (Loret et al., 2016; Lundqvist et al., 2011; Monopoli et al., 2011). The pathological and functional responses of lung cells to particles are also highly dependent on the physicochemical properties of NPs in association with direct interactions of particles on the state and activity of each individual cell (Rothen-Rutishauser et al., 2008). Therefore, any changes of particle properties after addition in the cell culture medium can highly influences the NPs-induced cytotoxicity (Lenz et al., 2013; Monopoli et al., 2011; Stoeger et al., 2006). All the above-mentioned parameters of NPs may be altered in suspended form when used under submerged cell culture-conditions. This may lead to unreliable outcomes and significantly limit the reproducibility of the system for further investigation (Lee et al., 2009). One of the main limitation of cells cultured at submerged conditions is to assess of the exact particle dose available for interaction with the cells (Lenz et al., 2013). This is due to the lack of direct methods for calculating particle number from the hydrodynamic particle properties, like size, density and shape. Moreover, a substantial fraction of the particles remains in the liquid or are lost in the lateral walls of the culture vessel, thereby influencing the actual dose of particles as well as their interaction with the cells. AIR-LIQUID INTERFACE Exposure experiments to air pollutants (PM/NPs and gases) under submerged cell culture conditions do not accurately address cell-cell and cell-stimulant interaction apart from dosimetry-related issues (Ahmad et al., 2014; Clippinger et al., 2016; Gminski et al., 2010; Lenz et al., 2013). To overcome the drawback related to physiological relevance of human airway mucosa, more complex models including multiple cells cultured at ALI (Lee et al., 2009; Rothen-Rutishauser et al., 2008; Thai et al., 2005) combined with advanced exposure systems to ensure accurate dosimetry (Ji et al., 2017; Klein et al., 2013; Rothen-Rutishauser et al., 2008) have been developed. In vitro exposure systems that deliver aerosolized pollutants to the surface of the cells cultured at ALI are of particular importance (Paur et al., 2011). In most of the exposure systems developed till date, deposition of aerosolized particles on cells occurs through the processes of diffusion and/or gravitation. Due to technical limitations, the maximum achieved dose using these systems remain generally far too low compared with the corresponding ambient pollutant concentration. Several factors influence the successful development of ALI-models that include choice of cell line, source of primary cells, coculture systems, mimicking preexisting conditions like chronic bronchits, dosimetry and customized cell culture conditions (medium, supplements, labware [Clippinger et al., 2016; Rothen-Rutishauser et al., 2008]). ALI system is also considered as a feasible approach to implement the “3 R principle”—replacement, reduction, and refinement of animal usage in pulmonary toxicity studies. Cell Line-Based ALI Model Different cell types like immortalized respiratory cell lines (eg, BEAS-2B and 16HBE140o, Calu-3, A549, NCI-H441) have been used to develop ALI models representing the human pulmonary system (Clippinger et al., 2016; Haghi et al., 2014; Zscheppang et al., 2018). Cell lines are homogenous and offer several advantages such as easy accessibility and can be use at high passages. However, respiratory cell lines on the other hand also exhibit some distinct limitations, such as their ability to produce mucus and form tight junctions. Similarly, quality assurances of the cell lines are also dependent on the source organization. American Type Culture Collection (ATCC) and European Collection of Authenticated Cell Cultures are popular among researchers because of their quality control by standardized protocol (eg, storage) for the development and maintenance of the cell lines (Clippinger et al., 2016). The Calu-3 cell line is of human lung adenocarcinoma; derived from metastatic site origin, commercially available from the ATCC and displays epithelial morphology as well as adherent growth (Forbes, 2000; Rothen-Rutishauser et al., 2008). The presence of tight junctions and the secretory activity makes the Calu-3 cell line a promising tool for lung drug absorption studies. BEAS-2B cell line was first developed from normal human epithelium and have been used to study airway structure and function on regular basis, however this cells line does not form tight junction (Clippinger et al., 2016; Zscheppang et al., 2018). Similarly, 16HBE is also a normal human airway epithelial cell line, which form extensive tight junction, but having less mucus producing ability compare to primary bronchial epithelial cells (PBECs) (Rothen-Rutishauser et al., 2008; Zscheppang et al., 2018). To develop alveolar airway mucosa A549 and NCI-H441 cell lines are mostly used. A549 cell lines are derived from pulmonary adenocarcinoma, which is consequently characterized as being representative of the alveolar type II pneumocytes of the human lung and are one of the most commonly used cell line in toxicity studies (Braakhuis et al., 2015; Rothen-Rutishauser et al., 2008; Zscheppang et al., 2018). Although, A549 cells produce surfactant protein, but they are not able to develop tight junction (Rothen-Rutishauser et al., 2008; Elbert et al. 1999). Whereas, for ion transport studies NCI-H441 cells are better choice since those cells form polarized tight monolayers and express organic cation transporters with P-glycoprotein (Rothen-Rutishauser et al., 2008; Zscheppang et al., 2018). Many of the widely used cell lines (Calu-3, A549) are carcinogenic in origin and likely to have altered genetic and phenotypic characteristics compared with normal cells. Therefore, this has led to a more conservative view on the cell line’s for suitability to recapitulate the bronchial and/alveolar epithelial cells phenotype. Primary Cell-Based ALI Model The use of primary human/animal cells cultured at ALI is considered as a better condition than using cell lines. The toxicological findings on the basis of primary human cells at ALI condition are more acceptable to predict the regulatory assessment compared with primary animal cells (Gruber and Hartung 2004; Rothen-Rutishauser et al., 2008). The cells isolated from fresh animal tissue (primary culture) represent a more heterogeneous population of different cell types, hence isolation and identification of specific cell types need to be performed carefully during establishment of methodology (Zscheppang et al., 2018). Use of primary human lung cells is limited due to the availability of human pulmonary tissue and their (donor) variation. However, human bronchial epithelial cells from normal and diseased donors as well as human lung microvascular endothelial cells are available from commercial sources. Thai and co-workers (Kao et al., 2005; Thai et al., 2005) have developed ALI-models using both human and mice primary airway epithelial cells. The establishment of ALI-models is highly dependent on the use of animal-derived media supplements, such as bovine pituitary extract and growth factors. Another crucial factor for the development of physiologically relevant in vitro model is the differentiation of epithelium consisting of basal cells, ciliated cells, and goblet cells that are present in vivo (Zscheppang et al., 2018). To overcome the limitation of availability of primary cells, many researchers use the commercially available ALI-models. The human airway models (EpiAirway) developed by MatTek’s (MatTek Corporation, MA, USA), OncoCilAirTM and MucilAirTM (Epithelix Sarl, Suisse) are having more than 30 donors from healthy and diseased individuals (asthmatics, COPD patients, and smoker). These commercially available models can be used for toxicity screening, risk assessment, preliminary drug discovery studies and also the exploration of molecular pathways of specific disease (Zscheppang et al., 2018). The main advantage of the commercially available ALI-models is their long lifetime (3–12 months), allowing the possibility for repeated or chronic exposure studies. However, the commercially available systems are limited in terms of flexibility (cell manipulation, genetic manipulation) and customization (disease-specific donor for primary cells (Zscheppan et al., 2018). Coculture System Introduction of multicellular ALI-models are considered an advancement of cell-cell communications comparable with in vivo condition (Fröhlich and Salar-Behzadi 2014). Therefore, the in vitro multi-cellular airway mucosa ALI-models that include different cell types (epithelial cells, macrophages, endothelial cells, and/or dendritic cells) are considered to be with high physiological relevance (Klein et al., 2013). Exposure of ALI-models to aerosolized PM or gaseous pollutants allows the direct interaction of particles and gases with cells surface representing in vivo situation (Ahmad et al., 2014; Gostner et al., 2016; Holder et al. 2008; Jing et al., 2015). In this aspect, another important physiological phenomenon is the formation of the thin liquid lining layer with surfactant mucus and the ciliary movement that is important for mucociliary clearance. Both surfactant and mucociliary clearance plays crucial role in particle displacement and retention (Möller et al., 2008; Rothen-Rutishauser et al., 2008). Another advanced multicellular ALI-model including A549 human blood monocyte-derived macrophages and dendritic cells has been developed by Rothen-Rutishauser et al. (2005), The A549 cells required to be cultured for adequate time at ALI to be able to produce surfactant. An alveolar ALI system consisting of 4 different human cell lines representing cell-cell response of the alveolar surface have been developed by Klein et al. (2013) to study the effect of aerosolized NPs. In this system endothelial cells (EA.hy 926) were seeded on the basolateral side of a micro porous membrane. Epithelial cells (A549) together with innate immune cells (mast cells: HMC-1 and macrophage-like cells: THP-1) were seeded on the apical compartment of the transwell inserts. These cocultures were designed in order to develop the 3D-organization of the cells, resembling the structure of alveolar barrier. Comparison of inflammatory and oxidative stress response between A549 cell lines and primary human PBECs cultured at ALI following exposure to CuO NPs under identical exposure concentration was performed. A549 cells were found to be more susceptible to CuO NP-mediated toxicity compared with PBEC (Jing et al., 2015). However, in order to mimic more closely the in vivo situations and to maximize the human relevance of in vitro testing, the combinations of complex cellular in vitro models with precise dosimetry calculations following exposure to aerosolized materials are in urgent need (Holder et al. 2008; Loret et al., 2016). Diseased ALI Model Ji et al. (2017) have recently shown the successful development of both normal and chronic bronchitis-like ALI-models using human PBEC. Treatment of PBEC with interleukin 13 (IL-13) at ALI condition resulting chronic bronchitis-like mucosa due to the generation of more mucus producing cells, a characteristic feature of chronic bronchitis. The study compared the effects of NPs exposure on both normal and chronic bronchitis-like diseased airway mucosa. These physiologically relevant ALI-models in combination with an exposure system that allows delivery of dry aerosolized NPs similar to inhalation exposure of humans (Ji et al., 2017; Zanoni et al., 2016). Similarly, several research groups have developed tumor spheroid models to study tumor growth, drug testing, in which a cluster of cells are grown together to form a viable tumour-like 3D structure (Zanoni et al., 2016; Zscheppang et al., 2018,). Thai et al. (2005) have developed ALI-models using both human and mouse primary epithelial cells to study the effect of IL-13 on 2 biomarkers, MUC5AC and hCLCA1/Gob5, which are frequently associated with surface mucous and goblet cells in asthmatic airways. However, development of ALI-model is time consuming and the availability of the primary cells from human donors is limited. This is coupled with a high variation of interindividual response to stimulant (Figure 2). Figure 2. View largeDownload slide Advancement of in vitro exposure model from submerged monoculture to physiologically relevant ALI model in association with exposure technique or exposure system to different environmental stimulants. Figure 2. View largeDownload slide Advancement of in vitro exposure model from submerged monoculture to physiologically relevant ALI model in association with exposure technique or exposure system to different environmental stimulants. Direct Comparisons of Particle Exposure in Submerged and ALI Culture Conditions Lenz et al. (2013) have investigated the cellular response of human alveolar epithelial-like cells (A549) cultured simultaneously at ALI and under submerged condition after exposure to airborne zinc oxide (ZnO) NPs. The findings of the study showed that cells exposed to ZnO-NPs at ALI system depicts significantly consistent cellular response in terms of oxidative stress and inflammation compared with submerged cultures. This observation is comparable with other literature data suggested that in vitro toxicity screening of NP with ALI-models may produce less false negative results than screening with submerged cell cultures (Lenz et al., 2013; Wilkinson et al., 2011). Culturing of PBECs at ALI followed by exposure to palladium nanoparticles (PdNPs) showed low uptake (Ji et al., 2017) in contrast to exposing the PBECs to PdNPs under submerged condition, where particles uptake was observed within 1 h after addition to the cultures (Wilkinson et al., 2011). The lack of particles in ALI-models could be explained by an existing mucociliary clearance. Direct Comparisons of Gaseous Air Pollutant Exposure in Submerged and ALI Culture Conditions Chronic or long-term exposure to gaseous air pollutants may be responsible for the onset of long-term respiratory effects like asthma, allergy and even onset of neurological disorders (Salthammer and Bahadir, 2009). Formaldehydes, carbon monoxide and ozone are the most commonly detected compounds in indoor environment and are also responsible for the development of acute toxicity (respiratory irritation; Petry et al., 2004; Salthammer and Bahadir, 2009). However, the major challenge in traditional submerged in vitro model is the available exposure system to expose the cells to gaseous or vaporized stimulants (Tsoutsoulopoulos et al., 2016). In most of the studies, toxicity assessment of gases is performed by direct addition of test compound (in liquid form) to the cell culture medium. Similar to the particles, the addition of chemicals in the medium may alter the properties of the stimulant due to interactions and binding with various components of the medium thereby resulting in unreliable outcomes (Gminski et al., 2010). Therefore, to test the cellular effect of gases in the gaseous phase, advanced in vitro ALI-model in combination with exposure systems capable of gaseous exposure are recommended (Dwivedi et al., 2018). However, there are only few exposure systems that are commercially available to suit the purpose allowing exposure of cultured cells to gas with a precise dosimetry and without any interfering medium (Gostner et al., 2016). The study by Ahmad et al. (2014) compared the variabilities in toxicity assessment of chlorine by using submerged and ALI-models. They demonstrated that chlorine reacts rapidly with aqueous surfaces to form hydrochloric and hypochlorous acids; and therefore, under submerged condition the observed toxicity effect might only demonstrated the effects of by-products such as hydrochloric acid rather than chlorine itself. Whereas, exposure of differentiated ALI cultures of human airway epithelial cells to chlorine gas allows a direct interaction of chlorine with cell surface in the absence of aqueous media. This is comparable to realistic exposure scenarios through inhalation of different gases in environmental and occupational settings. In a recent study (Dwivedi et al., 2018), ALI-models (including differentiated human PBEC together with fibroblasts) combined with a newly developed exposure system were exposed to gaseous form of acrolein, crotonaldehyde and hexanal. In this study we detected significant induction of inflammatory response was detected in association with elevated oxidative stress following exposure of human PBEC cultured at ALI to 3 different aldehydes (acrolein, crotonaldehyde, and hexanal) in gaseous form. Addition of these aldehydes directly to the cell culture medium resulted in nonsignificant alteration of inflammatory or oxidative stress response. The inflammatory response observed in ALI-models corresponded well with previous in vivo studies. This indicates that lung cells cultured at ALI are more suitable than the submerged in vitro model for toxicity assessment studies following exposure to aldehydes. The exposure chambers used in this study for exposing PBEC cultured at ALI to the aldehydes in gaseous form has been developed in our laboratory (Dwivedi et al., 2018). The steady target concentration of aldehydes was achieved by injection of an appropriate amount of aldehyde in the tedlar bag and the aldehyde was sucked in with clean air at a high flow rate into the exposure chamber. The actual concentration of aldehyde was monitored in real time using an attached gas chromatography just prior to each exposure (Dwivedi et al., 2018). The toxicological evaluation of tobacco whole-smoke (TWS) has been carried out with the primary focus on particulate fraction, which is often collected by passing TWS through a filter pad (Weber et al., 2013). The particulate fraction constitutes only 5%–10% of the total TWS generated from burning tobacco (Keith and Tesh, 1965). Hence the remaining compounds of TWS remain unassessed for toxicity evaluation. With the advancement of technology, such problems have been significantly resolved by preparing aqueous extracts of TWS by bubbling through a biological buffer or cell culture medium. However, due to the complexity and chemical composition, many compounds present in TWS remains undissolved in the biological buffer or in cell culture medium (Azzopardi et al., 2015; Thorne and Adamson, 2013). Therefore, in order to completely assess toxicological effects of TWS in the lung, an ideal in vitro cell culture system in association with an exposure system capable of delivering TWS in gaseous form including the particle fraction to lung cells exposed at ALI condition is required. Schamberger et al. (2015) has shown that chronic exposure of PBEC at ALI to cigarette smoke extract (CSE) through basal media significantly impair the cellular composition of the airway epithelium with reduced number of ciliated cells and increased number of club and goblet cells. Moreover, chronic exposure of PBEC to CSE extract has been associated with reduced cilia length and reduction in total acetylated tubulin (Schamberger et al., 2015). ALI-models used in this study are physiologically the most relevant in vitro models, and provides a suitable tool to study in detail the cellular effect observed in individuals with chronic lung diseases. However, exposure of these models to cigarette smoke instead of CSE might have been more appropriate to compare with the observed effects in vivo. Extensive developments of both in vitro ALI-models and the exposure system to deliver tobacco smoke have been observed during the last few years. The combination of these 2 criteria may finally allow to develop a well-defined uniform deposition system for TWS aerosol on pulmonary cells cultured in physiologically relevant in vitro models. Azzopardi et al. (2015) have shown such a combination by using Borgwaldt RM20S smoking machine combined with bronchial epithelial cell line (NCI-H292) cultured at ALI. In this study the investigators have demonstrated the sensitivity of epithelial cells line (NCI-H292) cultured at ALI by measuring cytotoxicity and inflammatory response following exposure to TWS and tobacco vapor (Azzopardi et al., 2015). The authors have shown the advantages of ALI-models in combination with this advanced exposure system in order to assess the sensitivity response of TWS derived from 2 different smoking regiments (Health Canada Intense and International Organization for Standard). AVAILABLE AIR POLLUTANT (PARTICLES AND GAS) EXPOSURE SYSTEM FOR ALI MODELS Many methods have been used to expose cells cultured at ALI to aerosolized stimulant either in dry powder form or by nebulization of solutions. The most crucial part of these exposure systems is to ensure the homogenous and even distribution of particles over the whole cell surface and reduced agglomeration of particles to avoid their alteration of physicochemical properties. Many research groups assessed the effects of air pollutants using diffusion exposure chambers. However, exposure of ALI-models to aerosolized particles or gases need to be further developed with easy and precise dosimetry system. The exposure systems developed specifically for ALI-based cell culture systems have been used to test several toxic substances such as different gases, copper NPs, carbon NPs, ZNO NPs, gold NPs, polystyrene NPs, cerium oxide NPs, and laser printer emission particles (Fuchs, 2002; Imanishi et al., 2009; Lenz et al., 2009). But standardized quantification of the deposited aerosols is essential as aerosols or NPs contained in the aerosol can be retained by components of the exposure systems or may form agglomeration which may result in undesired interaction and causing limitations of the outcomes. The most commonly used commercially developed in vitro particle exposure systems are from VITROCELL and CULTEX. In both these systems where the cells co-cultured at ALI and aerosolized particles or gas exposure takes place for 15–60 min under humid atmosphere at a temperature of 37°C (Gervelas et al., 2007; Niwa et al., 2007). Particle deposition is mostly driven by sedimentation and/or diffusion. Other established and commercially available in vitro aerosol exposure systems like CULTEX (Cultex Laboratories GmbH, Hannover, Germany) and electrostatic aerosol in vitro exposure system uses electrostatic precipitation of aerosol over the cells cultured at ALI condition (Aufderheide and Mohr, 2000; Niwa et al., 2007; Ritter et al., 2003). Exposure of ALI model to aerosolized air pollutants by these devices are technically easy to perform, but the deposition rates in these devices exhibit considerable variation. The obtained variation in deposition rate is probably related to the aerosolization technique (Sorokin 1970). Among the above described exposure systems, the major challenge however are concerning the formation of particle agglomeration with long exposure duration. Loading large amount of exposure materials (particles/gases) may also pose a technical hurdle, while minimal amount (1 mg) of particle loading is linked to the accuracy of the analytical balance and to achieve the target dose with a specific time duration (Brandenberger et al., 2010; Sorokin, 1970). MicroSprayer IA-1 C aerosolizer, a very small number of NPs are required to reach the target dose and the system shows less electrostatic interaction between particles and devices thereby represent a low risk of particle aggregation. However, the major disadvantage of this exposure system is the formation of droplets which may affect the homogeneous deposition of particles on in vitro model (Sorokin, 1970). Other noncommercial exposure systems with a limited number of users have been also described. These include the Biological aerosol trigger, Voisin chamber, Minu cell system, Nano Aerosol Chamber In vitro Toxicity, air-liquid interface cell exposure (ALICE) system. However, these exposure systems need further modification to standardize the particle deposition and dosimetry. ALICE system is an aerosol exposure instruments that are specially developed for exposure to aerosolized NPs, and has also been used by several investigators (Ritter et al., 2003). In ALICE exposure system, a dense cloud of droplets is generated which are finally transported to the exposure chamber with a specific flow rate. The generated droplets deposit uniformly over the cell surface due to single particle sedimentation by a process called cloud settling. ALICE exposure system allows a uniform, efficient and dose-controlled deposition of NP suspensions onto the cell surfaces at ALI; however the particle loading in suspension form may have an effect on the physicochemical characteristics of particles as already discussed (Fuchs, 2002). In spite of this apparent limitation, this particular exposure set-up might be considered as an optimal system to screen toxicity of NPs used in pharmaceutical industries where suspension-based aerosolized NP are used in the drug formulations; whereas in environmental or occupational exposure scenarios dry aerosolized NP are a more realistic exposure route (Duret et al., 2012). Recently, Ji et al. (2017) used an advanced exposure system (XposeALI) for exposure of ALI-models to dry aerosolized NP (Ji et al., 2017). The XposeALI system offers the scope for exposing ALI-models to dry aerosolized environmental pollutants. In this exposure system, aerosolized NPs are mainly generated by pushing small batches of dry NP powder through a high-pressure aerosol generator into a 300-ml holding chamber of the exposure system. Finally, the generated aerosol is pumped from the holding chamber through a Casella light dispersion instrument and dispersed uniformly into the exposure manifold, at a main flow rate of 120 ml/min. The main flow is diverted into 3 consecutive branch flows for individual exposure of 3 cell culture inserts at the time of exposure (Ji et al., 2017). This particular commercially available system is suitable for exposure of ALI cell culture inserts to aerosolized NPs, and it is also possible to compare and validate the in vitro findings with in vivo inhalation (rodents) exposure by using the same instrument but with different exposure modules. However, most of these exposure systems exhibit considerable variations in deposition rates, which might be due to different aerosolization technique and may also relate to particle properties (eg, size, carbon NPs, or metal NPs, etc). Therefore, further development of all of the discussed exposure system is required to avoid variation in deposition rate with particle properties. FUTURE DEVELOPMENT OF IN VITRO MODEL AND EXPOSURE SYSTEM The enormous effort made by many investigators have resulted an extensive advancement of the in vitro models and its usage at different levels of research covering toxicity screening to detailed molecular signaling pathway studies (Figure 2). Due to the emergence of nanotechnology and release of NPs in the environment from different sources (like automobile exhaust and abrasion of car tyres), there is an urgent need to understand the details of the NP- human body interaction and the plausible molecular mechanism for health risk assessment. In vitro models and particularly ALI-models mimicking human airways are of valuable support for the toxicological assessments of air pollutants. Further development of these systems is required to investigate cellular and adaptive responses through long term or chronic exposures and combined exposure studies of both NPs and gases. Additionally, ALI models of different levels of the lung (bronchial vs alveolar mucosa) as well as other organs (ALI model of gut or skin) need to be developed for further risk assessment. In the current review we have highlighted the different airway mucosa models and the corresponding exposure systems available commercially and noncommercially. Each of those exposure systems are unique in their own way and may require standardization at the next level to deliver both NPs and gases even with more precise dosimetry for in vitro studies. In vitro model is an important platform to screen cellular pathogenesis of air pollutant-mediated lung diseases; however, in vitro system lacks the complexity of the entire organism. Therefore, investigation and validation of current in vitro findings with in vivo outcomes, which includes detailed studies of air pollutants (particles and gases) induced toxicity and cellular responses in animal models (eg, rodents) using same exposure system are warranted. However, most of the exposure systems mentioned in this review are mainly developed for in vitro exposure studies. Whereas, XposeALI, is one of the commercially available platform that offers both in vitro and in vivo exposure to dry aerosolized NPs under identical exposure conditions.The validation of in vitro findings compared with in vivo outcomes will significantly enhance the strength of the in vitro data. This will allow researchers to perform cross-platform comparisons for further progression of translational research and will be the most important feasible approach to implement 3R strategy of animal model to perform air pollutant mediated pulmonary toxicity studies. 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Toxicological SciencesOxford University Press

Published: Mar 9, 2018

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