4.2. Isolation and Preconditioning of Bone-Marrow-Derived MSCs

Mice were euthanized by cervical dislocation. Bone marrow cells were obtained by flushing out femurs with PBS. Cells were cultured in a density of 1.5–2 × 10⁶/cm² in an MSC culture medium (Pan-Biotech, Aidenbach, Germany) supplemented with 2.5 ng/mL human basic fibroblast growth factor FGF (FGF-b, Peprotech, Hamburg, Germany), 100 U/mL penicillin, 10 µg/mL streptomycin (Sigma Aldrich, St. Louis, MA, USA) at 37 °C until a proliferative and homogenous MSC population was obtained. MSCs were characterized phenotypically by flow cytometry and by their potential to differentiate into chondrocytes, adipocytes and osteoblasts [18]. Bone marrow-derived MSCs of passage 9 were used in this study.

For preconditioning, MSCs were kept in an MSC culture medium supplemented with 2.5 ng/mL FGF-b until cells reached a 70–80% confluency. MSCs were stimulated with 30 ng/mL recombinant murine IFN-γ (Peprotech, Hamburg, Germany) and 3 ng/mL recombinant murine IL-1β (Peprotech, Hamburg, Germany) for 24 h. MSCs without cytokine treatment served as control. Then, cells were washed twice with PBS to remove any residual factors and serum and further cultured in RPMI 1640 medium (Pan-Biotech, Aidenbach, Germany) supplemented with 100 U/mL penicillin and 10 µg/mL streptomycin (Merck, Darmstadt, Germany). Twenty-four hours later, culture supernatants were centrifuged to remove cellular debris. Culture supernatants from three different MSCs cultures (with or without preconditioning) were pooled, aliquoted and stored at −80 °C until further use. In some experiments, pooled culture supernatants were concentrated ~4-fold by a centrifugal filter device (3 kDa cutoff; Thermo Fisher, Waltham, MA, USA) and stored frozen at −80 °C. Pooled MSC-CM or concentrated MSC-CM, respectively, were used for the cell culture experiments.