Bionoc 2

For exosome production, MenSC, UCMSC and fibroblasts between passage 4 and 8 were expanded in traditional 2D adherent cell culture and then seeded on BioNOC II micro carriers (CESCO Bioengineering, Taichung, Taiwan). For seeding, 8 × 10⁶ cells resuspended in 50 milliliters (ml) of DMEM with 10% FBS, 1% L-glutamine and 1% P/S and layered on top of two grams (g) of sterilized BioNOC II in a filter cap 250 ml Erlenmeyer flask (CLS431144, Sigma-Aldrich, Missouri, USA). The cells were maintained in static incubation during the first 16 hours (h) and were then supplemented with additional 200 ml of medium. For further culturing, the cells were maintained under constant agitation at 20 rounds per minute (rpm) on a rocking shaker (SK-R1807-E, DLAB Scientific, Beijing, China) in a humidified incubation chamber at 37 °C with 5% CO₂. To assess cell confluence, five micro carriers were removed each day, washed with phosphate buffered saline (PBS) three times and the cell nuclei were stained with Hoechst solution 1:2,000 for 10 minutes (min) (63493, Sigma Aldrich). When the cells reached 70% confluence on the carriers, they were washed three times with 250 ml of PBS and their medium was changed to FBS-free, phenol red-free DMEM (17–205-CV, Corning) containing 1 mM L-glutamine and 1% P/S. After 72 h the supernatants were collected for exosome purification.