Triple t175 flasks

For harvesting the conditioned medium (CM) for EV isolation, 3 × 10⁶ hBMSCs (passage 4) were cultured in growth medium in triple T175 flasks (ThermoFisher, Dreieich, Germany) until 80% confluency before onset of osteogenic differentiation. To induce osteogenic differentiation of hBMSCs according to established protocols [32 (link),34 (link)], the expansion medium was changed to osteogenic differentiation medium (α-MEM (Sigma-Aldrich, Steinheim, Germany), 10% FCS^depl-uc or 10% regular FCS, 4 mM GlutaMAXTM-I (Gibco, Paisley, UK), 1% penicillin–streptomycin (P/S), 10 μM ascorbic acid-2-phosphate, 10 mM ß-glycerophosphate and 100 nM dexamethasone (all from Sigma-Aldrich, Steinheim, Germany). Osteogenic differentiation was terminated after a maximum of 35 days (medium was replaced every 3 days).

Before onset of osteogenic differentiation, 3 × 10⁶ hBMSCs (passage 4) were seeded in triple T175 flasks (#132867; ThermoFisher, Dreieich, Germany) and cultured in growth medium until 80% confluency. To induce osteogenic differentiation according to established protocols [23 (link),24 (link)], the expansion medium was exchanged with osteogenic differentiation medium with the following composition: α-MEM (Sigma-Aldrich, Steinheim, Germany), 10% fetal calf serum (FCS) (Sigma-Aldrich, Steinheim, Germany) or EV-depleted FCS (FCS^depl-uc), 1% penicillin–streptomycin (P/S) (Sigma-Aldrich, Steinheim, Germany), 4 mM GlutaMAXTM-I (Gibco, Paisley, United Kingdom), 10 µM ascorbic acid-2-phosphate (Sigma-Aldrich, Steinheim, Germany), 10 mM β-glycerophosphate (Sigma-Aldrich, Steinheim, Germany) and 100 nM dexamethasone (Sigma-Aldrich, Steinheim, Germany). Osteogenic differentiation was conducted for up to 35 days, and culture medium was replaced every 3 days.