BMSC Harvest and Culture

Bone marrow was aspirated from a 2-year-old sheep (weight, 65 kg) under general anesthesia. We aspirated 10 mL of bone marrow (2 × 5 mL from different locations) from the posterior iliac crest into a 20-mL sterile polypropylene tube containing 10,000 IU of preservative-free heparin sodium (Wockhardt UK Ltd). The aspirate was combined with standard growth media, which was high glucose Dulbecco’s modified Eagle medium (Invitrogen), 10% fetal calf serum (ThermoFisher), and 1% penicillin-streptomycin (ThermoFisher). The aspirate was plated directly onto T-150 culture flasks and cultured at 37°C in a 5% CO₂ incubator. After 72 hours, the flask contents were discarded and washed with phosphate-buffered saline to remove nonadherent cells, and fresh growth medium was added to supplement the cells that had attached to the flask. When 90% confluent, the cells were detached using 0.25% trypsin-EDTA (Gibco). The BMSCs were subcultured, and the growth medium was changed every 3 days. The 10 million allogenic cells from passage 3 were used in the surgical procedures to provide approximately 2 million cells in the graft and 4 million cells per bone tunnel.