Inhalation is the most relevant entry point for nanoparticles (NPs) into the human body. To date, toxicity testing of nanomaterials in respect to oral, dermal and inhalative application is mainly based on animal experiments. The development of alternative test methods is the subject of current research. In vitro models can help to investigate mechanistic aspects, as e.g. cellular uptake or genotoxicity and might help to reduce in vivo testing. Lung cell lines are proper in vitro tools to assess NP toxicity. In respect to this, various cell models have been developed during the recent years, but often lack in a proper intact barrier function. However, besides other important in vivo criteria which are still missing like e.g. circulation, this is one basic prerequisite to come closer to the in vivo situation in certain mechanistic aspects such as particle translocation which is an important task for risk assessment of nanomaterials. Novel developed in vitro models may help to investigate the translocation of nanomaterials from the lung. We investigated the barrier function of the recently developed human lung cell lines CI-hAELVi and CI-huAEC. The cells were further exposed to CeO2 NPs and ZnO NPs, and their suitability as in vitro models for toxicological investigations was proven. The obtained data were compared with data generated with the A549 cell line. Measurement of transepithelial resistance and immunohistochemical examination of tight junctions confirmed the formation of a functional barrier for both cell lines for submerged and air–liquid cultivation. For particle exposure, hAELVi and huAEC cells showed comparable results to A549 cells without losing the barrier function. CeO2 NP exposure revealed no toxicity for all cell lines. In contrast, ZnO NPs was toxic for all cell lines at a concentration between 10–50 μg ml−1. Due to the comparable results to A549 cells CI-hAELVi and CI-huAEC offer new opportunities to investigate nanoparticle cell interactions more realistic than recent 2D cell models.

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

Dermal replacement materials bioactivated with cyanobacteria have shown promising potential for wound regeneration. To date, extraction of cyanobacteria RNA from seeded scaffolds has not been described. The aim of this study was to develop a method to isolate total RNA from bioactivated scaffolds and to propose a new approach in determining living bacteria based on real-time PCR. Transgenic Synechococcus sp. PCC 7002 (tSyn7002) were seeded in liquid cultures or scaffolds for dermal regeneration in vitro and in vivo for 7 days. RNA was extracted with a 260/280 ratio of ≥2. The small subunit of the 30S ribosome in prokaryotes (16S) and RNAse P protein (rnpA) were validated as reference transcripts for PCR analysis. Gene expression patterns differed in vitro and in vivo. Expression of 16S was significantly upregulated in scaffolds in vitro, as compared to liquid cultures, whilst rnpA expression was comparable. In vivo, both 16S and rnpA showed reduced expression compared to in vitro (16S: in vivo Ct value 13.21 ± 0.32, in vitro 12.44 ± 0.42; rnpA in vivo Ct value 19.87 ± 0.41, in vitro 17.75 ± 1.41). Overall, the results demonstrate rnpA and 16S expression after 7 days of implantation in vitro and in vivo, proving the presence of living bacteria embedded in scaffolds using qPCR.