The Panc-1 cell line was obtained from the ScienCell (USA). Cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium, Thermo Fisher Scientific, USA) supplemented with 10% EV-depleted fetal bovine serum (obtained via ultracentrifugation at 160,000 g for 12h) (Thermo Fisher Scientific, USA). Cells were incubated at 37 °C with 5% CO2.
Preparation of sEVs-containing medium
Before sEVs isolation, the supernatant from Panc-1 cells was handled as previously described20. Briefly, supernatant was centrifuged at 300 g for 10 min to remove cells, centrifuged at 2,000 g for 10 min to remove the dead cells, and then centrifuged at 10,000 g (Thermo Fisher ST 16R, USA) for 1h to remove the cell debris and microvesicles21. The remaining supernatant was sEVs-containing medium.
Preparation of crude sEVs
The following methods were separately used for preparation of crude sEVs (Figure 1A): (1) Ultracentrifugation (UC): 50 ml of sEVs-containing medium was centrifuged at 100,000 g (SW32Ti, Beckman Coulter XPN-100 Ultracentrifuge, USA) for 70 min at 4 °C, and the pellet was re-suspended in 200 μL of PBS 21; (2) Ultrafiltration (UF): 50 ml of sEVs-containing medium was concentrated using a 100 kDa ultrafiltration centrifugal tube (Merck Millipore, USA), PBS was added to re-suspend the concentrate, resulting in a final volume of 200 μL 22; (3) Co-precipitation (Co-P): 50 mL of 8% PEG 6000 (Solarbio, China) solution was prepared and then mixed with 50 ml of sEVs-containing medium at 4 °C for 12 hours, and then centrifuged at 4,000 g for 60 min. The pellet was re-suspended in 200 μL of PBS. Crude sEVs were stored at -80°C (within 2 days.) before further purification.
Purification of sEVs
Based on the results of evaluation of pellets after concentrating sEVs, ultracentrifugation was used for concentrating sEVs for further purification of sEVs. Four methods were used for purifying crude sEVs (Figure 1B): (1) Density gradient ultracentrifugation (DGUC): 200 μL of crude sEVs was loaded onto the top of a 12 mL discontinuous sucrose (Solarbio, China) gradient solution (15%, 20%, 25%, 30%, 40%, 60% sucrose in PBS, 2 mL for each gradient solution) and then centrifuged at 100,000 g for 16 h at 4 °C. 12 fractions (1 ml) were collected for each gradient 23. (2) Size exclusion chromatography (SEC): Sepharose CL-2B (Solarbio, China) was loaded into an injector (10 mL), with cotton blocked at the bottom. 200 μL of crude sEVs was loaded onto the top of the column. Then the column was eluted by PBS, and each 1mL of eluate was collected for 12 sequential fractions 24. (3) UC: 200 μL of crude sEVs was washed in PBS. Then the solution was centrifuged at 10,000g, 4 °C for 70 min. The pellet was re-suspend by 200 μL of PBS. (4) Immunoaffinity capture (IAC): Magnetic beads (BeaverBeadsTM Protein A/G immunoprecipitation kit, Beaver, China) were washed and then activated by incubating with 100 μL of anti-CD63 antibody (50 μg/mL, ab134331, abcam, UK) for 15 min. After magnetic separation, the anti-CD63-conjugated beads were incubated with 200 μL of crude sEVs at 25 °C for 1 h. The sEVs-beads complexes were separated by a magnet and eluted and re-suspended in 40 μL of PBS. Purified sEVs were stored at -80°C within two days before analysis.
Analysis of fraction from DGUC and SEC
Total protein level in each fraction was determined by using a BCA Protein Assay Kit (MultiSciences Biotech Co., China). The level of CD63 (CSB-E14107h) and CD9 (CSB-EL004969HU) in each fraction was determined by using an enzyme-linked immuno-sorbent assay (ELISA) kit (CUSABIO Biotech Co. Ltd., China). After analysis, fractions 6, 7, 8, 9, 10 from DGUC were mixed used for further analysis, while fractions 9, 10 from SEC were mixed and used for further analysis.
Characterization and analysis of crude sEVs and purified sEVs
Nanoparticle Tracking Analysis (NTA)
For crude sEVs, samples were diluted 100 times with PBS before NTA. For purified sEVs, samples obtained via IAC was diluted 125 times, samples obtained via SEC was diluted 2.5 times, samples obtained via UC was diluted 25 times, while samples obtained via DGUC was not diluted before NTA (NS300, Malvern, UK). The RR of particles was calculated as the following formula. The assay was repeated 3 times.
Transmission Electron Microscopy
10 μL of samples was dropped onto an ultrathin carbon film-coated 400 mesh copper grid and washed with PBS for two times. After drying excess liquid, the EVs-coated grid was stained by phosphotungstic acid (1%) and then washed with PBS for two times and then dried and imaged with a multipurpose field emission transmission electron microscope (TEM, JEM-1200EX, JEOL Ltd., Japan).
The protein level was determined by using a BCA Protein Assay Kit. The Protein Recovery rate (RR) of the purification process was calculated as the following formula. The assay was repeated 3 times.
5× SDS-PAGE Loading Buffer (New cell and Molecular Biotech Co., Ltd, China) was added into the sample. The sample was kept at 100 °C for 10 min. 6.02×109 EVs were loaded on each well in 12% SDS-PAGE (Lianke Bio, China). After the electrophoresis, the proteins were transferred to PVDF membrane (Millipore, USA). The membrane was blocked with 5% milk solution for 1.5 h, followed by incubation with anti-CD63 (ab134045, abcam, UK), anti-CD81 (GB111073, Servicebio, China), anti-CD9 (ab92726, abcam, UK), anti-CD47 (ab108415, abcam, UK), anti-GAPDH (AF7201, Affinity, China), and anti-ago 1(#9388, Cell Signaling, USA). The PVDF membrane was washed and then incubated with Peroxidase-conjugated Goat anti-Rabbit IgG (ZSGB-Bio, China). The membrane was incubated with ECL luminescent (New cell and Molecular Biotech Co., Ltd, China) for 3 min for detection.
Coomassie brilliant blue staining
5× SDS-PAGE Loading Buffer (New cell and Molecular Biotech Co., Ltd, China) was added into the sample. The sample was kept at 100 °C for 10 min. 6.02×109 EVs were loaded on each well in 12% SDS-PAGE (Lianke Bio, China). After the electrophoresis, the gel was incubated with 20 mL of working solution (0.0025% Coomassie brilliant blue, 45% methanol, 10% glacial acetic acid) (Solar bio, China) for 1 h, and then washed by elution solution (25% methanol, 8% glacial acetic acid) for 4 h.
The level of CD63, CD81, TSG101, beta-actin, GAPDH, CD47 (CUSABIO Biotech Co. Ltd., China) and ago-1 (MyBiosource, Canada) in purified samples and in crude sEVs were determined by ELISA kits. The protein per EV was calculated by the following formula. The assay was repeated 3 times.
Digestion of proteins
SDT solution (4% SDS, 100 mM Tris-HCl, pH 7.6) was added into the purified sEVs. The sample was incubated under boiling water for 15 min, followed by centrifugation at 14,000 g for 15 min. The supernatant was collected as protein sample. DTT (Sigma, USA, 43819-5G) was added in the protein sample to 100 mM. The sample was incubated under boiling water for 5 min, and then cooled to room temperature. 200 μL of UA buffer (8M Urea, 150mM Tris-HCl, pH 8.5) was added to the sample, followed by centrifuging at 12,500 g for 15 min using a 30 kDa ultrafiltration tube and centrifuged at 12,500 g for 15 min. Then, 100 μL of iodoacetamide (IAA) buffer (100mM IAA in UA) was added and kept at room temperature in darkness for 30 min. The sample was centrifuged at 12,500 g for 15min. 100 μL of UA buffer was added to the supernatant and then centrifuged at 12,500 g for 15 min again. 100 μL of 40 mM NH4HCO3 solution was added to the sample followed by centrifugation at 12,500g for 15 min. Then, 40 μL of Trypsin buffer was added (4μg Trypsin in 40 μL of 40 mM NH4HCO3) to the sample and incubated at 37°C for 16-18 h. The sample (in a filtration tube) was centrifuged at 12,500 g for 15 min, then 20 μL of 40 mM NH4HCO3 solution was added and centrifuged at 12,500 for 15 min to obtain the filtrate. A C18 cartridge (WAT023590, Waters) was used to desalinate. After freeze-drying, 40 μL of 0.1% methanol solution was added to the solid to re-solute the sample.
Then sample was separated by Easy nLC (Thermo Fisher Scientific, USA). Solution A: 0.1% FA; Solution B: 0.1% FA, 80% ACN. Chromatographic column (Acclaim PepMap RSLC 50 μm × 15 cm, nano viper, P/N164943, Thermo Fisher Scientific) was balanced by 100% solution A. Velocity of flow was 300 nL/min. Gradient elution: 0-5 min, solution B 3%; 5-45 min, solution B 3%-28%; 45-55 min, solution B 28%-38%; 50-55 min, solution B 38%-100%; 55-60 min, solution B 100%.
The sample was analysed by Q Exactive (Thermo Fisher Scientific, USA). Analysis time was 1 h. The mode was positive ion mode. Range of parent ion was 350-1800 m/z. The resolution of mass spectrometry was 7000. AGC target was 3e6. 1 stage Maximum IT was 50 ms. Via full scan 10 MS2 scans were acquired. MS2 Activation Type was HCD. Isolation window was 2 m/z. The resolution of 2 stage mass spectrometry was 17,500. Microscan was 1. 2 stage. Maximum IT was 45ms. Normalized Collision Energy was 27eV.
Data-dependent acquisition was performed. The peptides database was Uniprot_HomoSapiens_20386_20180905, downloaded in http://www.uniprot.org. MaxQuant 184.108.40.206 was used to qualitative analysis. Label Free Quantitation was used for quantitative analysis.
Data were presented as mean values ± SD. One-way analysis of variance (ANOVA) and students’ t test were performed at the significance level α = 0.05.