Lsgs supplemented medium 200

HUVECs migration was analyzed using xCELLigence real-time cell analysis (RTCA). Cell migration measurements were carried-out using 16-well CIM-plates (Agilent, Reference 5665817001). CIM-plates have upper and lower chambers for each well separated by a microporous membrane (in polyethylene terephthalate) with pore size of 8 µm and in contact with microelectrodes. Briefly, 160 µL per well of LSGS-supplemented Medium 200 (Gibco) were deposited in lower chambers. CIM-plates were then assembled under the cell culture hood using CIM-Plate 16 assembly tool. Subsequently, 25 µL/well of LSGS-supplemented Medium 200 (Gibco) were added in upper chambers to cover the membrane surface, and CIM-Plates were incubated for 1 hour at 37°C, 95% humidity and 5% CO₂. Background measurement step was then performed (every minute during 10 min). HUVECs cell suspensions (100 μL and 1 × 10⁴ viable cells/well) containing or not EVs (4 × 10⁷ EV/well) were then added in upper chambers. Each condition was performed in duplicates with a programmed signal detection schedule once every 10 s during 24 h (early effects), then every 10 min for 48 h (late effects). Control cells were only incubated with LSGS-supplemented Medium 200 (Gibco). Results are expressed using normalized cell index (NCI) as described by Bourgois et al. (2019) (link).

Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco, 100 µL/well) was coated onto 24-well plates and cultured in a 37°C for 30 min to allow matrix gel polymerization. HUVECs (Gibco) were removed from culture after 7 days in LSGS-supplemented Medium 200 (Gibco), trypsinized and resuspended in LSGS-supplemented Medium 200. HUVECs (5 × 10⁴ cell/well) were seeded into each well in LSGS-supplemented Medium 200 with MSC-EVs samples (2 × 10⁸ EV/well). After 12 h incubation, the cells were stained with 2 μg/mL of Calcein, AM (Invitrogen, Waltham, MA, United States) incubated for 30 min at 37°C. Fluorescence images were captured at a ×4 magnification using a CKX53 brightfield and epifluorescence microscope (Olympus, Allentown, PA, United States). The quantification of angiogenic network formation was performed by counting the number of nodes, junctions, segments and calculating the total segment length based on the collected images, utilizing the NIH ImageJ analysis software, as described in (Gilles et al., 2012 ).