Vivaflow 200 cell

Extracellular vesicles were isolated from sweet oranges of the species Citrus sinensis (CS-EVs). Fresh fruits were purchased from a local producer (certified origin), carefully washed and manually squeezed. The orange juice (350 ml obtained by squeezing approximately four oranges) was mixed with an equal volume of 5% glucose solution and filtered through a paper filter and then through a 0.45-μm membrane filter. To remove residual debris, the juice was finally centrifuged at 800 g for 20 min. To concentrate the juice, the supernatant was ultrafiltered by Vivaflow 200 cell (Sartorius) and ultracentrifuged at 100,000 g for 3 h. The pellet obtained after the ultracentrifugation was washed one time with PBS and ultracentrifuged for additional 3 h. The washed pellet was resuspended in PBS, and protein content was determined by Pierce™ BCA Protein Assay Kit (Thermo) according to the manufacturer's protocol. CS-EVs were finally stored at −20°C until use.

CCS of transfected HEK 293 cells was harvested and filtered with a 0.22 µm filter. Then, CCS was submitted to ultra-centrifugation (20 min-17,000× g at 4 °C) using Optima L-80 XP Ultracentrifuge (Beckman Coulter). The resulting supernatant was ultrafiltrated using a Vivaflow 200 cell with a cut-off of 100,000 Da (Sartorius, VF20H4) and finally concentrated to 1 mL. Purification of exosomes has also been performed using size exclusion chromatography (SEC) on ÄKTA Pure™ chromatography system (GE Healthcare, Uppsala, Sweden) equipped with a 1 mL loop and a F9-C fraction collector. The 1 mL concentrated supernatant was injected on a double tandem Superose 6 Increase 10/300 GL column [55 (link)]. Unicorn software 7.0 was used for data collection and analysis. During SEC, absorbance at 260–280 nm, conductivity, and pH were monitored. Fractions of 1 mL were harvested and processed for further studies.