3D FloTrix miniSpin bioreactor (Beijing CytoNiche Biotechnology Co. Ltd., Beijing, China) is used for scalable microcarrier-based 3D dynamic culture of hucMSCs, and it mainly includes a miniSpin agitator and spinner flasks of three volumes. hucMSCs were seeded to a density of 5 × 104 cells/mL in basal medium supplemented with 4% supplement medium; the speed of the miniSpin agitator was set to 50 rpm/min, and cells were cultured at 37 °C in a 5% CO2 incubator. During the cell culture, cell viability was determined using calcein AM and propidium iodide (PI) kit (Wako, Japan) according to the manufacturer’s instruction.
RAW264.7 cells (peritoneal macrophages of mice) could be activated and release some inflammatory mediators. These substances may impair pulmonary tissues and stimulate fibroblast (NIH-3T3 cells) proliferation and differentiation into myofibroblasts.
RAW264.7 cells were treated with 50 μg/mL silica for 24 h, and the supernatant was collected. NIH-3T3 cells (lung fibroblasts of mice) were maintained with the original silica supernatant for 24 h and harvested for future experiments. See additional detail in online supplement.
Isolation and identification of hucMSC-Exos
Serum-free medium of the 3D culture was centrifuged at 3000g for 15 min to remove dead cells and cellular debris. Exoquick exosome precipitation solution (System Biosciences, Palo Alto, USA) was added to the ultrafiltrate, mixing thoroughly by inversion. Exosomes were stored at − 80 °C or used in downstream experiments. See additional detail in online supplement.
Transmission electron microscopy
hucMSC-Exos were resuspended with PBS at a concentration of 100 μg/mL. Then, the mixture was applied to the copper grid and fixed with 2% phosphotungstic acid negatively. A transmission electron image was acquired using an HT-7700 transmission electron microscope (Hitachi, Tokyo, Japan).
Nanoparticle tracking analysis
In order to quantify hucMSC-Exos and size distribution, NanoSight instrument (ZetaVIEW, Germany) and ZetaView 8.04.02 software were used for nanoparticle tracking analysis (NTA). For NTA, 10 μL hucMSC-Exos was diluted to 20,000 times gradually and automatically calculating the size of at least 10,000 particles. The PBS was evaluated before the experiment to ensure that it was free of particles.
In vitro tracking
hucMSC-Exos were fluorescently labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil, Beyotime Biotechnology, Shanghai, China). The labeled exosomes were isolated through Exoquick exosome precipitation solution (System Biosciences, Palo Alto, USA) to remove excess dye. Mixtures were co-cultured with NIH-3T3 cells in DMEM for 4 h at 37 °C. Then, the cells were washed thrice with PBS to remove excess dye, co-cultured with Hoechst dye (Beyotime Biotechnology, Shanghai, China) at 37 °C for 1 h, and then observed under a fluorescence microscope.
Animal model of silicosis and ethics statement
A total of 60 C57BL/6J mice were randomly divided into three groups: the control (n = 20), silica (n = 20), and silica + hucMSC-Exos (n = 20). This study was approved by the Laboratory Animal Care and Use Committee at Capital Medical University (AEEI-2018-223) and abided by the principle of reduction, replacement, and refinement described in the National Institute of Health Guide for the Care and Use of Laboratory Animals. See additional detail in online supplement.
In vivo tracking
Mice were intravenously injected with hucMSC-Exo labeled with fluorescent 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR, Life Technologies, Carlsbad, CA, USA). The Carestream FX Pro imaging system (Bruker BioSpin MRI GmbH, Ettlingen, Germany) was used to capture the fluorescent signal at different time points (1, 6, 24, 48, 72, and 96 h) in the body. See additional detail in online supplement.
Lung function measurements
The flexiVent FX system (SCIREQ, Inc., Montreal, Canada) was used for respiratory function measurement. The system was equipped with an FX2 module and operated using the Flexi Ware v8.0 software. See additional detail in online supplement.
The samples of hucMSC-Exos were subjected to SDS-PAGE on 10% gels. The following primary antibodies were used: CD81 (1:1000, ab33697) and TSG101 (1:1000, ab125011) were purchased from Abcam (Abcam, USA) and CD63 (1:1000, 25682-1-AP) was procured from Proteintech (Proteintech, USA). The protein of NIH-3T3 cells was resolved on 8% SDS polyacrylamide gels. The primary antibodies, COL1A1 (1:100, sc293182) and FN (1:200, sc8422), were purchased from Santa Cruz (Santa Cruz, USA), and GAPDH (1:1000, 2118s) was obtained from Cell Signaling Technology (Cell Signaling Technology, USA). ECL detection reagent (Absin, Shanghai, China) was used to detect the signals, and the signals were imaged by Tanon-5200 system (Beijing Yuan Ping Hao Biotech, China). The quantification was calculated by ImageJ software to analyze the intensity of the gray scale images. See additional detail in online supplement.
RNA isolation and reverse transcription quantitative PCR (RT-qPCR)
The levels of gene expression were calculated by normalizing to the glyceraldehyde-3-phosphate dehydrogenase level. The COL1A1 sense sequence was 5′-GCTCCTCTTAGGGGCCACT-3′, and the antisense sequence was 5′-CCACGTCTCACCATTGGGG-3′. The FN sense sequence was 5′-CTATAGGATTGGAGACACGTGG-3′, and the antisense sequence was 5′-CTGAAGCACTTTGTAGAGCATG-3′. See additional detail in online supplement.
The left lung of mice was inflated overnight with 10% formalin. The organs were embedded in paraffin and cut into 5-μm-thick slices. The sections were observed using 3D histological technologies (Pannoramic Scan, Japan) with × 200 or × 400 magnification.
Pulmonary hydroxyproline assay
Pulmonary hydroxyproline (HYP) was detected using a conventional hydroxyproline colorimetric assay kit (Nanjing, China). Protein levels in the lung tissue were detected at an absorbance of 550 nm based on the manufacturer’s instruction, and all data were expressed in milligrams per gram of protein.
The sample of NIH-3T3 cells was fixed with 0.1% Triton X-100 and blocked with 3% BSA, and then incubated with primary antibodies against COL1A1 (1:100, sc293182) and FN (1:100, sc8422) overnight at 4 °C. The cell climbing piece was incubated with secondary antibodies conjugated to fluorescein isothiocyanate at room temperature for 1 h, which was counterstained with DAPI subsequently. The images were captured with a Leica TCS SP8 STED confocal microscope (Leica Microsystems, Germany).
Statistical analysis was performed by SPSS software version 19. All the data were presented as mean ± standard deviation (SD). Student’s t test was used for comparisons of two groups, and one-way analysis of variance (ANOVA) was used for multiple-group comparisons. Imagines were performed using GraphPad Prism software. Differences were considered to be statistically significant when P < 0.05.