Basic fibroblast growth factor (bfgf)

Bone marrow aspirates were obtained from patients undergoing spine surgery at the Inselspital Bern. The Swiss Human Research Act does not apply to research that involves anonymized biological material and/or anonymously collected or anonymized health-related data. Therefore, this project did not need to be approved by the ethics committee. General Consent which also covers anonymization of health-related data and biological material was obtained. BMSC isolation was performed via Ficoll density gradient centrifugation as described in Rothweiler et al. [43 (link)]. Human BMSC were cultured in BMSC medium consisting of Minimum Essential Medium α-modification (Gibco by Life Technologies, Waltham, MA, USA, #1200-063 + 2.2 g/L NaHCO₃), 10% FBS (Sera Plus, PAN-Biotech, Aidenbach, Germany, #3702-P121812), 1× P/S (Gibco by Life Technologies, Waltham, MA, USA, #15140-122), 5 ng/mL basic fibroblast growth factor (bFGF; Fitzgerald, Acton, MA, USA, #30R-AF015).

Bone marrow from 20 different anonymous human donors (range min 18 years, max 85 years, Average 57.71 ± 19.34 years) was harvested after informed consent (Ethical approval: Freiburg, EK-326/08). A known co-morbidity was considered an exclusion criterion. Fresh bone marrow was diluted 1:4 and layered on top of Ficoll, in a proportion of 2.6 ml of Ficoll per ml of undiluted marrow. After centrifugation at 500 g for 20 min, the mononuclear cell-containing interface was recovered, and cells were counted using the Cell Scepter 2.0 Automated Cell Counter (Millipore). Isolated cells were seeded at a density of 50,000 cells/cm² into 300 cm² tissue culture flasks in Minimum Essential Medium Eagle, Alpha Modification (α-MEM; Gibco, UK) containing 10% fetal bovine serum (Sera Plus, PAN-Biotec 3702-P12812 Aidenbach, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, UK), and 5 ng/ml recombinant human basic fibroblast growth factor (bFGF, Fitzgerald Industries International, Acton, MA, USA). Cells were maintained at 37°C in 5% CO₂, 85% humidity atmosphere. Medium was refreshed every 2nd day. After 4 days, non-adherent hematopoietic cells were removed to select the mesenchymal stromal cell (hMSC) population.

Chondrogenic differentiation was induced in human synovial derived stem cells isolated from synovial membrane (n = 4: male 42 y, male 41 y, male 19 y, male 54 y; obtained with full ethical consent approved by the Ethics Committee of the University of Freiburg as part of the “Tissue Bank for Research in the Field of Tissue Engineering” project (GTE-2002) and the biobank “Osteo” (AN-EK-FRBRG-135/14)). Samples were washed in 1:3 Betaisadona/phosphate-buffered saline (PBS) in Dulbecco’s modified Eagle medium containing 4.5 g/L glucose (DMEM-HG; Gibco, Thermo Fisher, Zürich, Switzerland) and then incubated in CollP-solution (1% collagenase) at 37 °C for 4 h.
After tissue digestion, the suspension cells were centrifuged at 500× g for 5 min, collected, and seeded in flasks for expansion with DMEM-HG.
hSDSCs were seeded at a density of 3000 cells/cm² in DMEM-HG containing 10% MSC-qualified fetal bovine serum (FBS) (Pan Biotech, Aidenbach, Germany), 100  U/mL penicillin, 100 μg/mL streptomycin (Gibco, Thermo Fisher, Zürich, Switzerland), and 5 ng/mL recombinant human basic fibroblast growth factor (bFGF, Fitzgerald Industries International, Acton, MA, USA). Cells were cultured at 37 °C in a 5% CO₂, 85% humidity atmosphere. Medium was changed every 2nd day until 70% confluence.