Dmi1 inverted phase microscope

Wells of a 96-well plate were coated with 50 μL of Matrigel (Corning) and incubated at 37°C for 30 min to allow Matrigel polymerization. Later, 20,000 HUVECs, re-suspended in the SF medium, were seeded on the top of the Matrigel and incubated at 37°C and 5% CO₂ for 6 h in the presence of different secretome fractions (CM, DM, mEVs, and sEVs) derived from 60,000 MSCs. Images were acquired using a Leica DMi1 inverted phase microscope, and analysis was performed on at least three different areas using the ImageJ Angiogenesis Analyzer plugin (Carpentier et al., 2020 (link)). Data were normalized to a positive control.

To evaluate MSC-derived CM, DM, and EV ability to induce HUVEC migration, a Transwell system (Corning, New York, United States) was used. Transwells, with a pore size of 8 μm, were placed in a 24-well plate, and 50,000 cells re-suspended in the SF medium were seeded inside the inserts. Secretome fractions (CM, DM, mEVs, and sEVs) derived from 150,000 MSCs of the different culture conditions were put in the lower chamber. After 9 h of incubation at 37°C and 5% CO₂, inserts were washed with PBS, and the non-migrated cells in the upper part of the membrane were gently removed using a cotton swab. Migrated cells were fixed in 10% buffered formalin for 10 min and stained with 1% methylene blue for 30 min. Images were acquired using a Leica DMi1 inverted phase microscope, and analysis was performed on at least five different areas using the ImageJ Cell Counter plugin. Data were normalized to a positive control.