Apoptotic Bodies

Vesicles were harvested using a differential centrifugation method commonly used for enriching exosomes16 (link), i.e.: conditioned media harvested from cell cultures was first centrifuged for five minutes at 500 g, to remove cells and larger debris. Media was then transferred to a new tube, and centrifuged at 2,000 g for ten minutes to remove cell debris and larger vesicles, such as apoptotic bodies. Large microvesicles were depleted by centrifugation at 10,000 g for 30 minutes. The media was then added to 35 mL polypropylene centrifuge tubes (Beckman Coulter Inc., #326823) in 35 mL aliquots, and centrifuged at 100,000 g (average relative centrifugal force) for 70 minutes using the SW-28 swing-bucket rotor in an Optima XL-100K ultracentrifuge (Beckman Coulter Inc.). Pelleted vesicles were suspended in 1 mL of PBS by gently vortexing, followed by agitating on an orbital shaker. The concentrated vesicle suspension was then centrifuged again in 1 mL polypropylene tubes (Beckman Coulter Inc., #347287) in an Optima MAX-E tabletop ultracentrifuge using a TLA120.2 rotor (Beckman Coulter inc.) to further purify the vesicles and facilitate re-suspension of the pellet in a small volume of PBS (50–100 μL). PBS was analyzed by NTA prior to suspension of vesicles and confirmed to contain particle numbers below the detection threshold of the instrument. The pellet was dissolved in particle free PBS by gentle vortexing followed by agitating on an orbital shaker for 15 to 30 minutes at room temperature until the solution was transparent and free of all aggregates. Samples were frozen at −80 °C until being further analyzed by nanoparticle tracking (see below) and protein quantification. All centrifugation steps were conducted at 4 °C. A subset of samples were processed as above, however, after centrifugation of the samples at 10,000 g, they were further centrifuged over a 30% sucrose cushion, as described by Thery et al., to enhance the purity of the enrichment16 (link). Briefly, four milliliters of sucrose solution was layered into 35 mL centrifugation tubes below a less-dense culture media layer. After centrifugation at 100,000 g for 75 min, 3.5 mL of the cushion (now containing the vesicles) was aspirated by piercing the tube and extracting with a 18-gauge syringe, carefully leaving any contaminants of greater, or lesser density. The 3.5 mL sucrose solution was then suspended in 31.5 mL PBS, and centrifuged a subsequent time at 100,000 g for 70 minutes to remove the sucrose. The purified pellet was re-suspended as described for the differential centrifugation procedure.

Cell-derived exosomes were isolated via an ultracentrifugation method using a Beckman L8-70M ultracentrifuge with an SW28 rotor (Beckman Coulter, Brea, CA, USA). The cell culture supernatant was collected in 50 mL centrifuge tubes and subjected to a sequential centrifugation procedure to clean up non-exosome components in the samples: (1) 500× g for 10 min to remove cell debris; (2) 10,000× g for 30 min to remove apoptotic bodies; and (3) 50,000× g for 60 min to remove micro-vesicles. The supernatant was carefully transferred to new tubes between each step, without disturbing the pellet. Finally, the supernatant was centrifuged at 100,000× g for 90 min to harvest the exosomes. The isolated exosomes were then imaged using an imaging flow cytometry system at the RPCI core facility. A Western blot analysis of exosome markers CD64 and FLOT1 was performed on isolated exosomes and whole cell lysates using the corresponding antibodies (Boston Bio-Products, Milford, MA, USA).

MSCs were cultured in complete medium to 80% confluence and then incubated with extracellular vesicle-free medium for 48 h. After 48 h, conditioned medium was collected and centrifuged at 1500 g for 30 min to remove apoptotic bodies and cell debris, followed by incubation with Ribo™ Exosome Isolation Reagent (for cell culture media, RiboBio, C10130-2) for 12 h at 4°C. The supernatant was centrifuged at 2000 g for 30 min. The supernatant was discarded, and the pellet was suspended in PBS (100 μl) and stored at -80°C.
The particle size and concentration of small extracellular vesicles were analyzed using nanoparticle tracking analysis (NTA). The collected small extracellular vesicles were fixed on carbon-coated copper grids with 1% glutaraldehyde and then stained with 1% phosphotungstic acid. The samples were examined using a JEM-2100 transmission electron microscope (TEM). And western blotting for CD63, CD81, and TGS101 was used to characterize the collected EMPA-sEV and MSC-sEV. To verify whether small extracellular vesicles could be absorbed by H9c2 cells, small extracellular vesicles were labeled with PHK26 and cocultured with H9c2 cells for 24 hours. And the cell nucleus were stained with DAPI after coculture.