The alveolar epithelial cell lines (NCI‐H1703 (ATCC, VA, USA) and NCI‐H441 (ATCC)) were cultured in a complete cell-growth medium, (RPMI‐1640 (HyClone, UT, USA)) supplemented with 10% fetal bovine serum (FBS; HyClone) and a 1% antibiotic/antimycotic solution (HyClone). Lung fibroblasts (MRC5 (ATCC)) were cultured in MEM α (HyClone) with 10% FBS and a 1% antibiotic/antimycotic solution. Lung microvascular endothelial cells (HULEC‐5a (ATCC)) were cultured in the MCDB 131 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10 mM l‐glutamine (Sigma‐Aldrich, MO, USA), 1 µg/mL hydrocortisone (Sigma‐Aldrich), 10 ng/mL human EGF recombinant protein (Thermo Fisher Scientific), 10% FBS, and a 1% antibiotic/antimycotic solution. All cell types were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The cells were subcultured upon growth to a sufficient level of confluence.
Engineering and 3D culture of the alveolar barrier
The alveolar barrier construct was fabricated by printing the four different cell types in a layer-by-layer manner using the drop‐on‐demand inkjet bioprinting system (Jetlab II; MicroFab, TX, USA). The total numbers of printed NCI‐H1703, NCI‐H441, MRC5, and HULEC‐5a cells were 8 × 104, 1.6 × 105, 1.8 × 105, and 3 × 105, respectively, which was consistent with the numbers of cells in the natural human alveoli . The all‐inkjet‐printed alveolar barrier was cultured in a 1:1:1 mixture of the complementary cell-culture medium (RPMI-1640, MEM α, and MCDB 131) with aprotinin (100 Kallikrein inhibitor unit) (A1153, Sigma‐Aldrich). The growth medium (500 µL) was added to the basolateral side of the 12‐mm cell culture insert, whereas the apical side was exposed to air during the incubation. The culture medium was replaced once a day. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 for 7 days before the dust-particle assessment. The information on detailed fabrication process can be described elsewhere .
Dust preparation and tissue exposure
As model fine dust particles, Arizona A2 fine test dust (ISO 12103-1, Powder Tech. Inc., MN, USA) was used which consists of 69-77% quartz. The particles were stirred in Dulbecco's phosphate‐buffered saline (DPBS, HyClone) at concentrations of 67.2, 224, and 672 μg/mL, after which they were vortexed for a few seconds before use. After 7 days of air exposure, the alveolar barrier constructs were treated with dust particles at concentrations of 30, 100, and 300 μg/cm2 in DPBS. To determine the concentration of dust particles per unit area, 500 μL of the solution was administered to the tissue epithelial layer over an area of 1.12 cm2. The epithelial layer of the control group was immersed in a 500 μL DPBS solution without dust. The dust-particle-exposure durations were 1, 3, 5, and 7 days. Over time, the dust particles settled in the epithelial layer.
Tissue proliferation assay
Tissue proliferation and metabolic activity were assessed in the dust-treated alveolar barrier constructs using Cell Counting Kit 8 (CCK 8; Dojindo Laboratories, Japan). The assay reagent was mixed with the cell growth medium in the ratio of 1:9. Thereafter, 0.5 mL of the mixture was added to the basolateral side of the 12-mm cell-culture insert and incubated for 1.5 h. The ultraviolet A (UVA) absorbance of the mixture was measured at 450 nm at each time point using a microplate reader (Spark; Tecan, Switzerland).
Measurement of tissue-barrier integrity
Warm (37 °C) DPBS portions (0.5 mL) were added to the apical side and the basolateral chambers (1.5 mL) of the cell culture insert. Thereafter, the alveolar barrier model was incubated for 20 min. The transepithelial/endothelial electrical resistance (TEER) was measured using an EVOM2 (World Precision Instruments, FL, USA) instrument with double chopstick electrodes (STX2, World Precision Instruments). a rubber O-ring was mounted on the cell culture insert to minimize the effect of contraction of a collagen basement membrane. The cell-free permeable membrane (reference) was also measured for the TEER while the O-ring was attached.
The permeability of the alveolar barrier model was investigated with 70 kDa FITC‐dextran (Sigma‐Aldrich) as a probe for successful permeability. The alveolar barriers were rinsed gently with Hanks’ balanced salt solution (HBSS; Sigma‐Aldrich) two times and transferred to another fresh 12‐well plate, which was filled with 1.5 mL of HBSS. The HBSS (0.5 mL) containing 1 mg/mL of FITC‐dextran was added to the apical side of the cell culture insert and incubated for 1 h. The concentration of the transferred FITC‐dextran from the basolateral chamber was determined using a fluorescence multi‐well plate reader (Spark; Tecan) with excitation and emission wavelengths of 490 and 520 nm, respectively. A new sample was used at each time point.
For the histological evaluation of the dust-treated alveolar barrier models, the samples were fixed in 1 mL of a 4% paraformaldehyde solution that was added to the tissue basolateral chamber overnight. The samples containing a porous membrane were cut out from the cell culture insert. The samples were embedded in optimal cutting temperature compounds (Leica Biosystems, Germany) and frozen at −80 °C. Serial 10‐µm‐thick sections were obtained using a cryostat (CM1860; Leica Biosystems). The tissue sections were stained with hematoxylin (Mayer's; Dako, CA, USA) and eosin Y (0.5% alcohol; Merck, Germany) to visualize and compare the structures of the tissues treated with dust particles. The sections were stained with Alcian blue (pH: 2.5; Sigma‐Aldrich) for observation of secreted surfactant, and nuclear fast red (Sigma‐Aldrich) stained the cell nuclei.
To determine the effect of the dust-particle treatment on the apoptosis in the alveolar barrier construct, the frozen sections were examined for the presence of apoptotic cells with fragmented DNA by the TdT‐mediated dUTP nick‐end labeling (TUNEL) assay using a commercially available kit (G3250, Promega, WI, USA), as detailed in the manufacturer's protocol. The cell nuclei were stained with propidium iodide (Sigma‐Aldrich).
To observe the existence and degradation of ECM, immunohistochemical analysis was performed on the tissue slices. A blocking solution was prepared by mixing 5% FBS, 3% bovine serum albumin (BSA; Thermo Fisher Scientific), and 0.1% Triton X‐100 (Sigma‐Aldrich) in PBS. The primary antibodies used in these protocols were rabbit anti-collagen IV (1:200, PA1-28534, Thermo Fisher Scientific) and rabbit anti-laminin (1:200, ab11575, Abcam, Cambridge, UK). The Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibody was used to visualize the targets (1:100, a11034; Thermo Fisher Scientific). The cell nuclei were stained with Hoechst 33342 (1:5000; Sigma‐Aldrich). For the histological analysis, all the bright field and fluorescence images were captured by a microscope (Ti‐S; Nikon, Japan)
RNA extraction and gene analysis
The tissues were washed with PBS and centrifuged. The total RNA was extracted from pellets using the RNeasy Mini Kit (Qiagen, Germany). Afterward, it was measured quantitatively using an ultraviolet–visible (UV–Vis) spectrophotometer (NanoDrop One, Thermo Fisher Scientific). The extracted RNA was reverse-transcribed using the oligo dT and high-capacity cDNA reverse transcription kit (Applied Biosystems, CA, USA), according to the manufacturer's protocols. Each cDNA was detected using a real‐time polymerase chain reaction (PCR) system (StepOne Plus, Applied Biosystems) with the SYBR Green Master Mix (Applied Biosystems). The sequences of the forward and reverse primer pairs are shown in Table S1, Supporting Information. The reference gene, glyceraldehyde 3‐phosphate dehydrogenase, was used to normalize the raw CT (cycle threshold) values.
Quantitative data are expressed as means, with error bars representing ± standard error of the mean. One‐way analysis of variance (ANOVA) with Tukey test was carried out to determine the statistical significance of the differences between experimental groups using the OriginPro 2016 software (OriginLab, MA, USA). The sample size (n) and preprocessing normalization of data are given in the corresponding figure legends. In all cases, a p-value of <0.05 was considered to reflect significance.