Qevoriginal columns

EVs were extracted from plasma samples by size exclusion chromatography method using the qEVoriginal columns (Izon Science). Every 0.5 ml of plasma yielded 1.5 ml of purified EVs in the 1xPBS buffer. EVs precipitation was performed overnight at +4 °C on a rotating mixer with L1CAM/CD171 Monoclonal Biotin Conjugated Antibody (eBio5G3 (5G3), Thermofisher) in the presence of 3% BSA and 3x Proteinase/Phosphatase Inhibitors cocktail (Sigma-Aldrich). After overnight incubation samples were additionally incubated with Pierce™ Streptavidin Plus UltraLink™ Resin (Thermofisher) for 1 h at +4 °C. As a washing step, sample tubes were centrifuged with 500 × g for 1 min, the supernatant was removed and saved as non-neuronal fraction of plasma EVs, and resin beads were washed 3x times with cold 1 ml of pre-filtered (with 0.22 μM filter) 1xPBS buffer. After final wash, precipitated neuronal-enriched EVs were eluted by incubation of the Streptavidin beads with 100 μl of Pierce™ IgG Elution Buffer (ThermoFisher) at room temperature with mixing for 5 min. As a negative control, EVs precipitation was performed with biotin-conjugated IgG2a kappa Isotype Control (eBM2a, ThermoFisher).

Size exclusion chromatography was performed using qEV original columns (for DNA-isolation; Izon Science) or qEV2 70 nm columns (for WB and TEM; Izon Science) according to the manufacturer’s instructions. After equilibration of the column, the recommended volume of clarified plasma was loaded onto the qEV columns and fractions 7–9 were eluted. The process was repeated for increased plasma volumes, with a column-washing step in between. Eluted fractions (containing sEVs) were combined and concentrated using Amicon® Ultra-15 Centrifugal Filter Units (100 kDa, Merck) and subjected directly to downstream analyses.

SS-hAFSC-conditioned medium was collected from ~ 5–10 × 10⁶ SS-AFSCs (2 × T225 flasks, 60 ml culture medium in total) cultured in serum-free conditions for the indicated time periods. SS-AFSC sEVs for tube formation assay and in vivo experiments were isolated from ~ 40–55 × 10⁶ SS-AFSCs (10 × T225 flasks, 300 ml culture medium in total). Conditioned medium was spun at 300 g for 10 min, 4 °C to remove dead cells. Supernatant was then centrifuged at 10,000 g for 40 min, 4 °C (polycarbonate tubes, 355,630, Beckman Coulter; MLA-55 rotor, Optima MAX-XP, Beckman Coulter) to remove cell debris and large vesicles. After discarding the pellet, conditioned medium was concentrated to ~ 200–450 µl using Vivaspin-15R ultrafiltration units (30 kDa, Hydrosart membrane, Sartorius). The remaining concentrate was immediately processed for SEC on qEVoriginal columns (iZON Science) [8 (link)]. Fractions were collected and pooled as indicated and stored at − 80 °C.

For sEV isolation from cell-culture media, approximately 50–75 mL was used. Media was centrifuged at 200 RCF for 5 min followed by centrifugation of the resulting supernatant at 720 RCF for 10 min. The samples were concentrated by Amicon Ultra-15 Centrifugal Filter Unit with a MWCO of 3 kDa (#UFC900324, Merck Chemicals and Life Science AB, Solna, Sweden) until reaching a volume of approximately 500 µL. sEV isolation was done by size-exclusion chromatography (SEC) on qEVoriginal columns (Izon Science, Oxford, UK) per manufacturer’s instruction. Briefly, columns were equilibrated with filtered PBS, and thereafter samples were gently added. Each sample was eluted in fractions of about 500 µL by adding PBS and fractions 6–10 were collected, pooled into a total volume of 2.5 mL, concentrated to 500 µL using Amicon^® Ultra-4 Centrifugal Filter Unit (MWCO = 3 kDa, #UFC800324, Merck Chemicals and Life Science AB), and subsequently subjected to nanoparticle tracking analysis (NTA) to verify particle size and amount. In an earlier study, sEV isolation by the same protocol was verified by scanning electron microscopy (SEM) showing the size and morphology of the isolated particles [45 (link)].