Isolation and Culture of Bone Marrow Stromal Cells

The isolation of BMSCs was performed following established protocols [23 (link),24 (link),25 (link)]. In short, mononuclear cells from donor bone marrow were extracted using a density gradient centrifugation protocol and seeded in 0.1% gelatin (Sigma-Aldrich)-coated T75 cell culture flasks (Sarstedt, Nümbrecht, Germany). Cells were cultivated in expansion medium (EM), consisting of DMEM high glucose, 12.5% v/v fetal calf serum (FCS), 2 mM L-glutamine, 1% v/v non-essential amino acids (NEAA), 50 µM β-mercaptoethanol (all Life Technologies, Darmstadt, Germany), 100 µg/mL penicillin/streptomycin (Sigma-Aldrich) and 4 ng/mL fibroblast growth factor 2 (Abcam, Cambridge, UK) at 37 °C and 5% CO₂ in a humidified (≥95%) atmosphere. A first medium exchange was conducted after 24 h to wash off non-adherent cells. Thereafter, medium exchanges were performed twice weekly. Cells were passaged at 80% confluency and transferred to liquid nitrogen storage until further use. The experiments were conducted with BMSCs in passage 4.