Patient-derived GCTB cells collection and cells culture

Patient-derived GCTB cells were isolated from tumor samples from tumor resections at the Changzheng Hospital as aforementioned. The tissue was mechanically cut into small pieces, and digested with 1.5 mg/ml collagenase B for 3 h at 37°C in DMEM containing 4.5 g/l glucose supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were collected by filtration (100-µm diameter filter), then centrifuged at 200 × g for 5 min at room temperature and washed twice in PBS. The cells were counted using a hemocytometer and resuspended at a density of 5×10⁵ cells/20 µl PBS. For morphological observation and TRAP staining, 1×10⁵ cells were seeded in 6-well plate and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in a 37°C humidified incubator with 5% CO₂ (30).

The hFOB1.19 cells were maintained in DMEM-F12 supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere with 5% CO₂ at 34°C. The medium was replenished every 2–3 days. After reaching 70–80% confluence, cells were passaged by treatment with 0.25% trypsin. Prior to intratibial injection, the cells were detached from the petri dish with 0.25% trypsin and centrifuged at 200 × g for 5 min at room temperature. Cells were resuspended into a 5×10⁵ cell/20 µl cell suspension in PBS.