Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface.

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules. Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

Validation of the CULTEX® Radial Flow System for the assessment of the acute inhalation toxicity of airborne particles.

The CULTEX® Radial Flow System (RFS) is a modular in vitro system for the homogenous exposure of cells to airborne particles at the air-liquid interface (ALI). A former pre-validation study successfully demonstrated the general applicability of the CULTEX® RFS and its transferability, stability and reproducibility. Based on these results, the methodology was optimized, validated and prediction models for acute inhalation hazards were established. Cell viability of A549 cells after ALI exposure to 20 pre-selected test substances was assessed in three independent laboratories. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity. Substances were considered to exert an acute inhalation hazard when viability decreased below 50% (prediction model (PM) 50%) or 75% (PM 75%) at any of three exposure doses (25, 50 or 100 µg/cm2). Results were then compared to existing in vivo data and revealed an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%. Depending on the applied PM, the within-laboratory and between-laboratory reproducibility ranged from 90 to 100%. In summary, the CULTEX® RFS was proven as a transferable, reproducible and well predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles.

Improvement of the CULTEX® exposure technology by radial distribution of the test aerosol.

The exposure of cellular based systems cultivated on microporous membranes at the air-liquid interface (ALI) has been accepted as an appropriate approach to simulate the exposure of cells of the respiratory tract to native airborne substances. The efficiency of such an exposure procedure with regard to stability and reproducibility depends on the optimal design at the interface between the cellular test system and the exposure technique. The actual exposure systems favor the dynamic guidance of the airborne substances to the surface of the cells in specially designed exposure devices. Two module types, based on a linear or radial feed of the test atmosphere to the test system, were used for these studies. In our technical history, the development started with the linear designed version, the CULTEX® glass modules, fulfilling basic requirements for running ALI exposure studies (Mohr and Durst, 2005). The instability in the distribution of different atmospheres to the cells caused us to create a new exposure module, characterized by a stable and reproducible radial guidance of the aerosol to the cells. The outcome was the CULTEX® RFS (Mohr et al., 2010). In this study, we describe the differences between the two systems with regard to particle distribution and deposition clarifying the advantages and disadvantages of a radial to a linear aerosol distribution concept.

Optimization of the CULTEX(®) radial flow system for in vitro investigation of lung damaging agents.

Exposure of the respiratory tract to airborne particles is gaining more and more importance due to the ubiquitous application of these particles in the field of industry, pharmacy and in daily life. Remarkably, the toxic properties and the underlying pathomechanisms with regard to inhalable substances have been insufficiently investigated so far. Thus, the EU Chemicals Regulation demands toxicological data (including the identification of potential inhalation hazards) for all chemicals placed on the market until 2018 (REACH). This requires extensive, technically complex and expensive inhalation toxicology studies that are usually generated in animal experiments. However, the legislation demands the consideration of the "3Rs" principle. Thus, in vitro-based test systems for the assessment of pulmonary toxicity are required. One promising approach to assess acute pulmonary toxicity of airborne particles is the CULTEX(®) RFS methodology that allows exposure of human lung epithelial cells at the air-liquid interface mimicking the alveolar situation. A prevalidation study showed the general applicability of this method. However, the clean air exposure group, which served as unexposed controls, exhibited some variations with regard to cell viability compared to the incubator control group. The aim of this study was therefore the identification of the possible causes and the improvement of methodological aspects. Several parameters including the general workflow, adjustment of airflow parameters, and cleaning procedures were investigated and adapted. Finally, our results showed the successful optimization of the CULTEX(®) RFS methodology for clean air exposure of A549 cells. However, although viability data in incubator controls and clean air exposures were equal, a distinct difference in cell morphology was observed that required further optimization. Additional experiments identified that open-wall cell culture inserts with a 2-fold pore density were found to be superior compared to the standard inserts and thus the deciding factor for the improvement of cell morphology. The presented findings are an important step in providing the CULTEX(®) RFS methodology as a promising alternative method to current in vivo testing in inhalation toxicology.

Direct exposure at the air-liquid interface: evaluation of an in vitro approach for simulating inhalation of airborne substances.

In toxicology, the strategies for testing the hazardous potential of substances are changing as a result of the ongoing progress in the development of in vitro methods and the demand of the authorities to reduce animal testing. Even in the complex field of inhalation toxicology with its high requirements on the technical implementation and cell culture models, the preconditions for using such methods are fulfilled. We here introduce a sophisticated technique that enables the stable and reproducible exposure of cultivated cells to airborne substances at the air-liquid interface by means of the CULTEX(®) Radial Flow System (RFS) module. The feasibility and suitability of the experimental setup is demonstrated by dose-response investigations of mainstream cigarette smoke and particulate matter of four substances in different lung epithelial cell lines. A dose-dependent cytotoxcity of the test substances was verified by applying different exposure times. The high reproducibility of the results indicate the reliability of the presented method and recommend the integration of such in vitro approaches in the field of inhalation toxicology by advancing their regulatory validation.

Use of the Cultex® Radial Flow System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparticles with special focus on the intra- and inter-laboratory reproducibility.

Exposure of the respiratory tract to airborne particles (including metal-dusts and nano-particles) is considered as a serious health hazard. For a wide range of substances basic knowledge about the toxic properties and the underlying pathomechanisms is lacking or even completely missing. Legislation demands the toxicological characterization of all chemicals placed on the market until 2018 (REACH). As toxicological in vivo data are rare with regard to acute lung toxicity or exhibit distinct limitations (e.g. inter-species differences) and legislation claims the reduction of animal experiments in general ("3R" principle), profound in vitro models have to be established and characterized to meet these requirements. In this paper we characterize a recently introduced advanced in vitro exposure system (Cultex® RFS) showing a great similarity to the physiological in vivo exposure situation for the assessment of acute pulmonary toxicity of airborne materials. Using the Cultex® RFS, human lung epithelial cells (A549 cells) were exposed to different concentrations of airborne metal dusts (nano- and microscale particles) at the air-liquid-interface (ALI). Cell viability (WST-1 assay) as a parameter of toxicity was assessed 24h after exposure with special focus on the intra- and inter-laboratory (three independent laboratories) reproducibility. Our results show the general applicability of the Cultex® RFS with regard to the requirements of the ECVAM (European Centre for the Validation of Alternative Methods) principles on test validity underlining its robustness and stability. Intra- and inter-laboratory reproducibility can be considered as sufficient if predefined quality criteria are respected. Special attention must be paid to the pure air controls that turned out to be a critical parameter for a rational interpretation of the results. Our results are encouraging and future work is planned to improve the inter-laboratory reproducibility, to consolidate the results so far and to develop a valid prediction model.

The CULTEX RFS: a comprehensive technical approach for the in vitro exposure of airway epithelial cells to the particulate matter at the air-liquid interface.

The EU Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) demands the implementation of alternative methods for analyzing the hazardous effects of chemicals including particulate formulations. In the field of inhalation toxicology, a variety of in vitro models have been developed for such studies. To simulate the in vivo situation, an adequate exposure device is necessary for the direct exposure of cultivated lung cells at the air-liquid interface (ALI). The CULTEX RFS fulfills these requirements and has been optimized for the exposure of cells to atomized suspensions, gases, and volatile compounds as well as micro- and nanosized particles. This study provides information on the construction and functional aspects of the exposure device. By using the Computational Fluid Dynamics (CFD) analysis, the technical design was optimized to realize a stable, reproducible, and homogeneous deposition of particles. The efficiency of the exposure procedure is demonstrated by exposing A549 cells dose dependently to lactose monohydrate, copper(II) sulfate, copper(II) oxide, and micro- and nanoparticles. All copper compounds induced cytotoxic effects, most pronounced for soluble copper(II) sulfate. Micro- and nanosized copper(II) oxide also showed a dose-dependent decrease in the cell viability, whereby the nanosized particles decreased the metabolic activity of the cells more severely.

Analytical in vitro approach for studying cyto- and genotoxic effects of particulate airborne material.

In the field of inhalation toxicology, progress in the development of in vitro methods and efficient exposure strategies now offers the implementation of cellular-based systems. These can be used to analyze the hazardous potency of airborne substances like gases, particles, and complex mixtures (combustion products). In addition, the regulatory authorities require the integration of such approaches to reduce or replace animal experiments. Although the animal experiment currently still has to provide the last proof of the toxicological potency and classification of a certain compound, in vitro testing is gaining more and more importance in toxicological considerations. This paper gives a brief characterization of the CULTEX® Radial Flow System exposure device, which allows the exposure of cultivated cells as well as bacteria under reproducible and stable conditions for studying cellular and genotoxic effects after the exposure at the air-liquid or air-agar interface, respectively. A commercial bronchial epithelial cell line (16HBE14o-) as well as Salmonella typhimurium tester strains were exposed to smoke of different research and commercial available cigarettes. A dose-dependent reduction of cell viability was found in the case of 16HBE14o- cells; S. typhimurium responded with a dose-dependent induction of revertants. The promising results recommend the integration of cellular studies in the field of inhalation toxicology and their regulatory acceptance by advancing appropriate validation studies.