Preparation of culture media and cell culture
EV-depleted serum (EDS) containing culture media (EDS-CM) were prepared at EDS (SBI) concentrations of 10%, 5%, 3%, 1%, 0.5%, and 0%, and normal complete culture medium (NC-CM) was prepared at a fetal bovine serum concentration of 10%. The culture media used were RPMI 1640 (GIBCO Company, USA). A549 cells used to produce sEVs were cultured in a 37℃ incubator containing 5% CO2 and examined for mycoplasma.
Collection of the culture supernatants
1×106 A549 cells were cultured in a 10 cm Petri dish (WHB) with NC-CM, and washed three times with PBS upon a cell density of 70%-80%, followed by replacement of 10 ml of EDS-CM. To determine the optimal concentration of EDS, the culture supernatants were collected 48 h after culture with the media containing 10%, 5%, 3%, 1%, 0.5%, 0% EDS.
In addition, to determine the number of days for continuous collection of supernatants, cell cultures were washed three times with sterile PBS (BI 02-024-1ACS) upon a cell density of 70%-80%, and the culture supernatants were collected daily for 5 consecutive days and 10 ml of fresh EDS-CM was replaced. (Fig. 1)
Observation of cell morphology and determination of cell viability and activity
Cell morphology was observed by inverted fluorescence microscopy (Nikon, Japan) after collection of supernatants and three washes of cell cultures with PBS. Specifically, trypan blue staining was performed after digestion of cells by trypsin (GIBCO, USA), and cell viability was measured by using an automatic whole-cell counter. The proliferative activity of 10%, 5%, 3%, 1%, 0.5%, and 0% EDS on A549 cells was tested using the CCK-8 kit (Beyotime, Shanghai, China). The 96-well plates (WHB) were plated with an equal number of cells, cultured with 100µL EDS-CM medium, and washed three times with PBS upon a cell density of 70%-80%. After 48 h culture with 100µL of EDS-CM, 10µl of the CCK-8 reagent was added to each well, followed by culture for 2 h in a 37℃ incubator containing 5% CO2. Absorbance was measured at 450 nm using a microplate reader (BioTek, USA).
sEV extraction from culture supernatants
The collected culture supernatants were centrifuged at 4℃ and 300 g for 10 min, and the supernatants were filtered through a 0.45 µm filter. The filtrate was centrifuged at 4℃ and 2000 g for 10 min, and the obtained supernatant was centrifuged again for at 4℃ and 10000 g for 10 min to obtain the supernatant free of cell debris, organelles and macromolecular particles of lipids and proteins.
Ultracentrifugation: 70 ml of the pretreated supernatant was transferred into a 70 ml centrifuge tube, centrifuged at 4℃ and 100000 g in an ultra-high speed centrifuge (Beckman, USA) for 70 min, and the EV-depleted supernatants were collected. The sEVs in the centrifuge tube were re-suspended in an appropriate amount of sterile PBS, then centrifuged for 70 min, re-suspended with 20 µL sterile PBS, and stored at -80℃ in the refrigerator (Thermo Fisher, USA).
Total Exosome Isolation kit (EI-kit) (Thermo Fisher, USA): 35 ml of the pretreated supernatant was transferred into a 50 ml centrifuge tube (CT-002-50A, LABSELECT, China), added with 17.5 ml of the reagent (Thermo Invitrogen 4478359), mixed well, and placed in a 4℃ refrigerator overnight (Thermo Fisher, USA). The centrifuge tube was then centrifuged at 4℃ and 10000 g for 1 h. The supernatant was discarded, and the sEVs were re-suspended with 10µL sterile PBS, and stored at -80℃.
Determination of protein concentration
The protein concentration of the pre-treated supernatant, EV-depleted supernatant and sEVs was measured using the BCA kit (Beyotime, Shanghai, China) by reading the absorbance at 562nm in a microplate reader.
SDS-PAGE analysis
All samples were mixed 4: 1 with 5X loading buffer (P0015, Beyotime, Shanghai, China) and boiled in a metal bath at 100°C for 10 min to denature the protein. Each sample was sequentially added to a 10% SDS-polyacrylamide gel (P0012AC, Beyotime, Shanghai, China) and subjected to electrophoresis at 80v for 30 min, and at 120v for 70min, and then the SDS-PAGE gels were stained with Coomassie brilliant blue (P1300, Beijing Solarbio Science & Technology Co., Ltd., China). The sEVs (20µL) obtained by ultracentrifugation of supernatants of each group was the experimental group, and the culture medium, EDS-CM, EDS, pretreated supernatant and EV-depleted supernatant were the control groups (20µL).
Transmission electron microscopy (TEM)
10µL of the sEVs suspension was dropped onto a copper mesh and allowed to precipitate for 1 min, the floating solution was sucked with a filter paper, then 10µL of uranyl acetate was dropped onto the copper mesh and allowed to precipitate for 1 min, and the floating solution was sucked and dried for 10 min at room temperature, and then imaged under Hitachi HT-7700 transmission electron microscope (Hitachi, Tokyo, Japan).
Nanoparticle tracking analysis (NTA)
The sample wells were washed with deionized water and then with 1X PBS buffer (BI, Israel), 10µL of the sEV suspension was diluted 1: 2,000 with 1XPBS, and tested on a ZetaView PMX 110 instrument (Particle Metrix, Germany) calibrated with 100nm polystyrene microspheres.
9.Flow cytometry detection (NanoFCM)
Fluorescence labeled sEVs obtained by ultracentrifugation were re-suspended in an appropriate volume of 1×PBS. After a performance test of the NanoFCM instrument passed using the standard substance, the samples were detected.
Western blotting analysis
Proteins were extracted from A549 cells with the RIPA and PMSF kits, and there was no need to extract sEV proteins. After the protein concentration of the samples was measured, 20 µg of each sample was mixed 4:1 (vol: vol) with 5X loading buffer, boiled for 10 min at 100°C in a metal bath, then added onto a 10% SDS-polyacrylamide gel, and subjected to electrophoresis first at 80v for 30 min and then at 120v for 70min. Proteins were transferred onto a PFDV membrane on ice at 200 mA for 60 min and incubated at room temperature for 30 min by using a rapid blocking solution (P0252-100ML, Beyotime, Shanghai, China). After washes with TBST, the membrane was incubated overnight with the primary antibody at 4℃. After washes with TBST, the membrane was incubated for 2h with the corresponding secondary antibody. Finally, the membrane was imaged with the developing reagent (17047, Zen-Bioscience, China) and developing instrument.
Co-culture of PKH26-labeled sEVs with A549 cells
The dye prepared according to the instructions for use (1µL of PKH26 + 9µL of Diulent C) (Umibio) was mixed well with sEVs, and the mixture was incubated for 10 min at room temperature in darkness, followed by addition of 1mL of PBS to stop the staining. The mixture was centrifuged at 100000 g for 17 min, the supernatant was discarded, and the sediments were re-suspended with sterile PBS. The centrifuge/re-suspend cycle was repeated three times. The stained sEVs were co-cultured with the cells for 24 h and then the cells were fixed with 4% paraformaldehyde and stained with DIPA and finally imaged under a laser confocal microscope (SP8, Leica Microsystems, Germany).
LC-MS/MS assays
3%- and 10%-sEV samples were removed from the refrigerator at -80℃ and determined for the protein concentration by the BCA method after adding a protease inhibitor for lysis. 3%- and 10%-sEV samples with the same amount of protein were digested with trypsin into peptides, which dissolved in mobile phase A and were subjected to gradient separation for 60 min at a flowrate of 450 nl/min in an ultra-high performance liquid chromatography system. The resulting peptides were ionized in the ion source at 1.75 kV and then analyzed by timsTOFpro mass spectrometry with a scan range of 100–1700 m/z. Data were acquired in the parallel cumulative serial fragmentation mode, and secondary mass spectrometry data were retrieved using Maxquant.
Statistical analysis
Data are expressed as mean +/- standard deviation. GraphPad Prism software was used for one-way ANOVA, test of the least significant difference between groups, and t test. All experiments were analyzed at least in triplicate and a p value < 0.05 was considered statistically different.