Development and Characterization of a 96-Well Exposure System for Safety Assessment of Nanomaterials.

In this study, a 96-well exposure system for safety assessment of nanomaterials is developed and characterized using an air-liquid interface lung epithelial model. This system is designed for sequential nebulization. Distribution studies verify the reproducible distribution over all 96 wells, with lower insert-to-insert variability compared to non-sequential application. With a first set of chemicals (TritonX), drugs (Bortezomib), and nanomaterials (silver nanoparticles and (non-)fluorescent crystalline nanocellulose), sequential exposure studies are performed with human lung epithelial cells followed by quantification of the deposited mass and of cell viability. The developed exposure system offers for the first time the possibility of exposing an air-liquid interface model in a 96-well format, resulting in high-throughput rates, combined with the feature for sequential dosing. This exposure system allows the possibility of creating dose-response curves resulting in the generation of more reliable cell-based assay data for many types of applications, such as safety analysis. In addition to chemicals and drugs, nanomaterials with spherical shapes, but also morphologically more complex nanostructures can be exposed sequentially with high efficiency. This allows new perspectives on in vivo-like and animal-free approaches for chemical and pharmaceutical safety assessment, in line with the 3R principle of replacing and reducing animal experiments.

Methodological considerations when conducting in vitro, air-liquid interface exposures to engineered nanoparticle aerosols.

Little consistency exists in the methodology for toxicological testing of aerosolized nanoparticles used in in vitro, air-interfaced culture (AIC) exposure systems for engineered nanoparticles (ENPs) risk-assessment, preventing inter-laboratory comparisons to identify dose thresholds for adverse effects. These inconsistencies result from heterogeneity in particle types, exposure durations, exposure systems, and dose metrics reported. We screened 10,241 studies in the literature for toxicological assessment of ENPs, resulting in 110 publications included after meeting eligibility criteria. In this review, we critically analyzed methodology within these studies to answer whether: (1) the administered dose or the deposited dose correlated better with biological response, (2) a difference existed between various AIC exposure systems when depositing the same dose, (3) consistent results were generated for nanomaterials with similar physico-chemical properties, (4) the deposited dose in vitro correlated to the deposited dose in vivo, and (5) AIC studies reliably modeled acute toxicity in vivo. Methods used in delivering, measuring, and reporting ENP aerosol doses in vitro are summarized. Dosimetry and biological response comparisons of AIC, conventional suspensions, and in vivo exposures are discussed through case studies on silver, zinc oxide, titanium dioxide, and multi-walled carbon nanotube exposures. Finally, based on these findings, recommendations are offered for design of future AIC experiments to aid standardization and comparisons of results.

A need to provide explanations for observed biological effects of radiofrequency exposure.

Although there is scientific consensus that radiofrequency (RF) exposure at high intensity can cause thermal effects, including well-established adverse health effects, there is still considerable controversy on whether low-intensity RF exposure can cause biological effects, especially adverse health effects. The objective of this paper is to describe several reported "non-thermal" effects that were later shown to be due to a weak thermal effect or an experimental artifact by properly conducted and thorough follow-on scientific research. First, the multiple factors that can cause different RF energy absorption in biological tissues are reviewed and second, several examples of experimental artifacts in published papers are described to demonstrate the importance of paying attention to dosimetry and temperature control. For example, isolated nerve response studies show that when temperature of the RF-exposed tissues is controlled, effects disappeared. During RF exposure, conductive electrodes routinely used in physiological studies have been shown to cause field intensification at the tips or contacts of the electrodes with biological tissue; thus, the RF exposure at the site of measurement could be much higher than the incident field. In some in vitro studies, a lack of temperature uniformity in RF-exposed cell cultures and rate of heating explain changes originally reported to be due to low-level RF exposure. In other studies, detailed dosimetry studies have identified artifacts that explain the reasons why so-called "non-thermal" effects were mistakenly reported. Researchers should look for explanations for their own findings, and not expect others to figure out what was the reason for their observed effects.

A localized ELF magnetic field exposure system for microscope cover-slips.

In extremely low frequency (ELF) magnetic field exposure systems for the inverted microscope stage where the cells grown on the entire microscope cover-slip are exposed to the magnetic field, the effects of variations in cell characteristics from one cover-slip to another on the experimental data cannot be readily identified. To overcome this drawback, a localized ELF magnetic field exposure system for cells grown on cover-slips was designed. The basic idea is to expose only a marked portion of the cover-slip to the magnetic field so that the effect of the ELF magnetic field on the cells grown on the same cover-slip can be observed under a microscope. A prototype system was built and tested. Experimental test results pertaining to the prototype system performance validate the proposed design approach. The paper concludes with a discussion of alternative approaches to the design of localized ELF magnetic field exposure systems.

Exposure systems for bioelectromagnetic experiments.

One of the most interesting questions in bioelectromagnetics is why there is a difference between results of experiments performed in various labs in "identical" conditions. One of the possible reasons is the difference of investigated objects, especially while performing experiments in vivo. However, the authors, as engineers, would like to focus readers' attention on the technical aspects of exposure systems, especially the presence and role of mutual interaction between biological objects under test (OUT) and the exposure system, the interactions between the objects, the role of polarization, the similarity of real exposure to that applied in experiments etc. All these factors may alter the results of experiments and lead to false conclusions.

The effect of airborne Palladium nanoparticles on human lung cells, endothelium and blood - A combinatory approach using three in vitro models.

A better understanding of the mechanisms behind adverse health effects caused by airborne fine particles and nanoparticles (NP) is essential to improve risk assessment and identification the most critical particle exposures. While the use of automobile catalytic converters is decreasing the exhausts of harmful gases, concentrations of fine airborne particles and nanoparticles (NPs) from catalytic metals such as Palladium (Pd) are reaching their upper safe level. Here we used a combinatory approach with three in vitro model systems to study the toxicity of Pd particles, to infer their potential effects on human health upon inhalation. The three model systems are 1) a lung system with human lung cells (ALI), 2) an endothelial cell system and 3) a human whole blood loop system. All three model systems were exposed to the exact same type of Pd NPs. The ALI lung cell exposure system showed a clear reduction in cell growth from 24 h onwards and the effect persisted over a longer period of time. In the endothelial cell model, Pd NPs induced apoptosis, but not to the same extent as the most aggressive types of NPs such as TiO2. Similarly, Pd triggered clear coagulation and contact system activation but not as forcefully as the highly thrombogenic TiO2 NPs. In summary, we show that our 3-step in vitro model of the human lung and surrounding vessels can be a useful tool for studying pathological events triggered by airborne fine particles and NPs.

Proof-of-Concept of Electrical Activation of Liposome Nanocarriers: From Dry to Wet Experiments.

The increasing interest toward biocompatible nanotechnologies in medicine, combined with electric fields stimulation, is leading to the development of electro-sensitive smart systems for drug delivery applications. To this regard, recently the use of pulsed electric fields to trigger release across phospholipid membranes of liposomes has been numerically studied, for a deeper understanding of the phenomena at the molecular scale. Aim of this work is to give an experimental validation of the feasibility to control the release from liposome vesicles, using nanosecond pulsed electric fields characterized by a 10 ns duration and intensity in the order of MV/m. The results are supported by multiphysics simulations which consider the coupling of three physics (electromagnetics, thermal and pore kinetics) in order to explain the occurring physical interactions at the microscopic level and provide useful information on the characteristics of the train of pulses needed to obtain quantitative results in terms of liposome electropermeabilization. Finally, a complete characterization of the exposure system is also provided to support the reliability and validity of the study.

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.

Uncertainity analysis of selected sources of errors in bioelectromagnetic investigations.

The aim of this paper is to focus attention of experimenters on several sources of error that are not taken into account in the majority of bioelectromagnetics experiments, and which may lead to complete falsification of the results of the experiments.

Assessment of cigarette smoke particle deposition within the Vitrocell® exposure module using quartz crystal microbalances.

BACKGROUND: Cigarette smoking is a cause of a variety of serious diseases, and to understand the toxicological impact of tobacco smoke in vitro, whole smoke exposure systems can be used. One of the main challenges of the different whole smoke exposure systems that are commercially available is that they dilute and deliver smoke in different ways, limiting/restricting the cross-comparison of biological responses. This is where dosimetry - dose quantification - can play a key role in data comparison. Quartz crystal microbalance (QCM) technology has been put forward as one such tool to quantify smoke particle deposition in vitro, in real-time. RESULTS: Using four identical QCMs, installed into the Vitrocell® mammalian 6/4 CF Stainless exposure module, we were able to quantify deposited smoke particle deposition, generated and diluted by a Vitrocell® VC 10 Smoking Robot. At diluting airflows 0.5-4.0 L/min and vacuum flow rate 5 ml/min/well through the exposure module, mean particle deposition was in the range 8.65 ± 1.51 µg/cm(2)-0.72 ± 0.13 µg/cm(2). Additionally, the effect of varying vacuum flow rate on particle deposition was assessed from 5 ml/min/well - 100 ml/min/well. Mean deposited mass for all four airflows tested per vacuum decreased as vacuum rate was increased: mean deposition was 3.79, 2.75, 1.56 and 1.09 µg/cm(2) at vacuum rates of 5, 10, 50 and 100 ml/min/well respectively. CONCLUSIONS: QCMs within the Vitrocell® exposure module have demonstrated applicability at defining particle dose ranges at various experimental conditions. This tool will prove useful for users of the Vitrocell® system for dose-response determination and QC purposes.

A review of in vitro cigarette smoke exposure systems.

In vitro test methods may be vital in understanding tobacco smoke, the main toxicants responsible for adverse health effects, and elucidating disease mechanisms. There is a variety of 'whole smoke' exposure systems available for the generation, dilution and delivery of tobacco smoke in vitro; these systems can be procured commercially from well-known suppliers or can be bespoke set-ups. These exposure technologies aim to ensure that there are limited changes in the tobacco smoke aerosol from generation to exposure. As the smoke aerosol is freshly generated, interactions in the smoke fractions are captured in any subsequent in vitro analysis. Of the commercially available systems, some have been characterised more than others in terms of published scientific literature and developed biological endpoints. Others are relatively new to the scientific field and are still establishing their presence. In addition, bespoke systems are widely used and offer a more flexible approach to the challenges of tobacco smoke exposure. In this review, the authors present a summary of the major tobacco smoke exposure systems available and critically review their function, set-up and application for in vitro exposure scenarios. All whole smoke exposure systems have benefits and limitations, often making it difficult to make comparisons between set-ups and the data obtained from such diverse systems. This is where exposure and dose measurements can add value and may be able to provide a platform on which comparisons can be made. The measurement of smoke dose, as an emerging field of research, is therefore also discussed and how it may provide valuable and additional data to support existing whole smoke exposure set-ups and aid validation efforts.