Extracellular vesicle isolation and purification

HeLa cells were seeded at 25,000 cells per cm² in a 100-mm cell culture dish. EV isolation and purification were conducted using complete medium (10 ml or 30 ml, depending on the experiment) obtained from cells at 70–80% confluency cultured for 16–48 h (see schematic representation in Fig. 2 A). Supernatants were obtained from cultures of cells with >95% viability. Cell culture medium was fractionated by centrifugation using the following steps: (i) 300 × g for 10 min (intact cells and cell debris), (ii) 2,000 × g for 20 min (apoptotic bodies), (iii) 10,000 × g for 40 min (large EVs/ectosomes/microvesicles). The supernatant of this last centrifugation was passed through a 0.2-μm filter (#83.1826.001; Sarstedt). Various concentrations of iodixanol-sucrose buffered solution were prepared by mixing homogenization medium and 50% of iodixanol-containing working solution. Homogenization medium was composed of 0.25 M sucrose (#S0389; Millipore Sigma), 1 mM EDTA (#RGC5135; KD Medical, Inc), and 10 mM Tris-HCl (#10708976001; Millipore Sigma) at pH 7.4. Working solution with 50% of iodixanol was prepared by mixing one part of iodixanol (OptiPrep, #07820; Stem Cells Technologies) with five parts of solution containing 0.25 M sucrose, 6 mM EDTA, and 60 mM Tris-HCl at pH 7.4. These buffering conditions maintain the integrity of EVs. Filtered supernatant (10 ml) was layered on top of a density cushion made up of 500 μl 10 and 50% iodixanol-sucrose buffered solution and centrifuged at 150,000 × g for 3 h in a SW41-Ti rotor (k-factor = 172). The interphase between 10 and 50% iodixanol-sucrose buffered solution contained crude exosomes. These crude exosomes were collected and subjected to further purification on a density gradient. Briefly, crude exosomes preparation (∼1 ml) was mixed with 3 ml of 50% iodixanol-sucrose buffered solution to a final concentration of 45% and layered at the bottom of the tube. Layers of 1 ml of 30, 28, 20, 10, and 0% iodixanol-sucrose buffered solution were successively overlaid and tubes were centrifuged at 120,000 × g for 18 h in a SW41-Ti rotor (k-factor = 214). Purified exosomes were collected from fractions 2 and 3 corresponding to ∼10 and 20% iodixanol-sucrose buffered solution and pooled for further analysis. Depending on the downstream application, 2 ml exosome suspension was either used directly or concentrated in 0.4 ml of PBS using centrifugation at 150,000 × g for 2 h in a TLA-100.3 fixed angle rotor (k-factor = 45).