title: Evaluation of a non-animal toolbox informed by adverse outcome pathways (AOPs) for human inhalation safety
design: Evaluation of a non-animal toolbox informed by adverse outcome pathways (AOPs) for human inhalation safety
library_construction_protocol: Following exposure to each test material for 12 days, EpiAveolar™ tissues were cut from the plastic unit, transferred into 2 mL Precellys tubes (Bertin Technologies, Montigny-le-Bretonneux, France) containing 700 µL of QIAzol lysis reagent and stored at -80°C until RNA extraction.  EpiAlveolar™ system was obtained from MatTek Corporation (cat. no. ALV-100-FT-MAC, ALV-100-FT-MAC-PE12). The tissue systems were derived from primary human alveolar epithelial cells, primary pulmonary fibroblasts, pulmonary endothelial cells, and cell line derived macrophages. These tissues were differentiated by the manufacturer prior to shipping to the testing facilities. Upon receipt, systems were maintained, for 2-7 days prior to use, at the air-liquid interface (ALI) in modified 6-well hanging top plates with 5 mL of EpiAlveolar™ culture medium (MatTek) in the basolateral compartment and 75 µL of media on the apical surface and incubated at standard culture conditions (37±1°C in a humidified atmosphere of 5% CO2 in air). Trans-epithelial electrical resistance (TEER) was measured and only systems with confirmed quality of the tight junction barrier (>300 Ω.cm2) and with approved morphological inspection were used in the experiments. To prepare the stock solutions to perform the exposures, the test materials were diluted in saline (AKEMI® Anti-Fleck Super/Akemi, Crystalline silica, Amorphous silica, Polyhexamethyleneguanidine phosphate/PHMG, LPS from Escherichia coli 055:B5, and Sulforaphane), ultrapure water (Doxorubicin) or DMSO (Amiodarone). PHMG and Akemi stock solutions were prepared daily before performing chemical exposures, whereas Crystalline and Amorphous silica suspensions were prepared via sonication for 1h and mixed by vortex prior to nebulization process; the same suspensions, stored at room temperature, were used for the whole 12-day experiments. Sulforaphane, Doxorubicin and Amiodarone stock solutions were filter sterilized and aliquots were stored at -20°C; fresh stock vials were used each day to avoid unnecessary freeze-thaws. After dissolving, LPS stock solution was stored at 4°C. The final DMSO concentration in the tissue-based assays did not exceed 0.1% (v/v in culture medium) for those test items in which DMSO was used as the vehicle; whereas where dilute saline is specified, this was a 1% (v/v) dilution in physiological saline (0.9%, w/v) and the final saline concentration in the tissue-based assays was 0.009%. Vehicle control groups were also tested in parallel. Except for Doxorubicin (which underwent a 6-day chemical exposure + 6-day without exposure), exposures were conducted daily, on 12 consecutive days with different concentrations, through aerosol (Akemi, Crystalline silica, Amorphous silica, PHMG, and Sulforaphane), apical (LPS) or basolateral (Amiodarone and Doxorubicin) liquid exposure methods. In the aerosol exposure method, the procedure was performed using a Vitrocell® Cloud 12 system chamber equipped with a quartz crystal microbalance (QCM). Each of the insert holders of the instrument base module was filled with Hanks' Balanced Salt solution (HBSS) or PBS prior to placing the tissue inserts. Then, each test item solution was placed into the nebulizer reservoir for aerosol material exposure. The nebulizer was activated until the material solution was consumed and discharged into the main exposure chamber and allowed to fully gravity deposit out of the Cloud 12 (determined using a QCM). After exposure, the tissues were placed back into their multi-well culture plates containing the same medium, a further aliquot of media was added apically (20 µL for Akemi, 75 µL for all others except LPS) and returned to incubator until next exposure. Media used during the 6- or 12-day experimental period was prepared without Supplement X by MatTek. In medium exposure, the apical or basolateral liquid of each tissue was removed and replaced with each test item medium solution and returned to the incubator until the next exposure. After the beginning of the exposure cycles, tissues were only re-fed with new culture medium every 3-4 days. Samples were homogenized followed by centrifugation at 12000 g at 4°C for 5 min. The supernatants were used to extract RNA through a combined semi-automated method using a Qiagen Rneasy 96 QIAcube HT kit. All samples were Dnase treated using a Thermo Fisher DNA-free kit, following manufacturer`s instructions. All these steps were performed by Laboratory 2 which, then, stored RNA samples in 96-well plates until shipment on dry ice with a temperature monitor to the RNA sequencing laboratory facility (Cambridge Genomics Services). TruSeq Stranded mRNA (Illumina) kit following manufacturer's protocol by Cambridge Genomics Services
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-14272:t3_liq_ctrl3_day8_1_s