Lm 10 nanoparticle tracking analysis system

For EV preparation, BM-MSCs and iPSC-MSCs at 70–80% confluence in 17% FBS αMEM medium were transferred to the chemically defined and protein-free medium based on CD-CHO medium (Invitrogen, cat. number 10743-002) as reported [20 (link)]. After 6 hr, the medium was discarded and replaced by fresh medium and recovered at 48 hr to isolate EVs. Briefly, the conditioned medium was centrifuged at 2565g for 15 min to remove cells and debris, and then EVs were isolated from the supernatant by ultracentrifugation at 100,000g for 90 minutes at 4°C using Sorvall WX Floor Ultra Centrifuge with AH-629 36 ml swinging Bucket Rotor (Thermo). EV pellets were dissolved in cold TM buffer (pH 8.6) overnight at 4°C and frozen at −80°C in TM buffer (pH 8.6) containing 1% sucrose and 1% glycerol. The size and concentration of EVs were analyzed using the NanoSight LM 10 Nanoparticle Tracking Analysis System (Malvern). The NanoSight instrument was calibrated with polystyrene latex 100 nm and 200 nm microbeads (NTA4088 and NTA4089). Samples were measured under a range of a particle count from 2 × 10⁸ to 1 × 10⁹ particles per milliliter.

For EV isolation, the conditioned medium was filtered to remove cellular debris (0.22 μm), and then EVs were isolated from the supernatant by ultracentrifugation (at 100,000 g for 16 h at 4°C) using Sorvall WX Floor Ultra Centrifuge with AH-629 36 ml swinging Bucket Rotor (Thermo Fisher Scientific, Waltham, MA). We pooled EVs from 8 to 10 cultures of MSCs (about 2.5 X 10⁶ cells per culture) into one sample and isolated EVs were resuspended with PBS at concentrations of 5 to 10 X10¹⁰/ml. The particle size and number of EVs were analyzed using the NanoSight LM 10 Nanoparticle Tracking Analysis System (Malvern, Malvern, UK). Also, the expression levels of EV surface markers CD9, CD63 and CD81 were analyzed by flow cytometry (CytoFLEX, Beckman coulter) using magnetic beads coated with anti-CD63 (human CD63 Isolation/Detection kit; Invitrogen), anti-CD63-FITC (clone H5C6; BD Biosciences), anti-CD81-PE (clone JS-81; Biosciences) and anti-CD9-FITC (clone eBioSN4; BD eBioscience). The isolated EVs were stored at -80°C.

To prepare fluorescently labeled nanovesicles and nanoghosts for in vitro assays, iPSC-MSCs (1 × 10⁶/mL) were labeled with 5 μl/mL DiI cellular membrane labeling-solution (ThermoFisher, D-282) for 20 min at 37 °C. For nanoghost preparation, iPSC-MSCs were hypotonically treated with tris-magnesium buffer followed by mild homogenization to make ghost cells without cytosol. Following the published protocols [7 (link), 8 (link), 23 (link)], ghost or intact iPSC-MSCs were extruded through 10, 5, 3, 1.2, and 0.4 μm polycarbonate membranes, and nanoghosts or nanovesicles were isolated by ultra-centrifugation for 45 min at 150,000×g at 4 °C. The sizes of these EV-mimics were analyzed using Nanosight LM 10 Nanoparticle Tracking Analysis System (Malvern). To examine the selective uptake of nanoparticles by PCa cells in vitro, 1 × 10¹⁰ DiI-labeled nanoghosts, nanovesicles, or liposomes (FormuMax, F60103F-DI) were incubated with 1 × 10⁵ PC3 cells, SMCs, or HUVECs in 1 ml DMEM for 15 min, 1 h, or 3 h, then cells were washed with phosphate-buffered saline (PBS) three times and analyzed by flow cytometry for DiI signal. The selective uptake of DiI-labeled nanoparticles by PC3 cells vs. SMCs or HUVECs was quantified as the log odds ratio (LOR) as reported [8 (link)].