4.2. Cell Culture Techniques

7F2 murine osteoblasts (American Type Culture Collection, Manassas, VA, USA, CRL-12557) were cultured in α-MEM media supplemented with 10 mM HEPES, 10% FBS, and 1% streptomycin and penicillin and maintained in a humidified, 37°C, 5% CO₂/air incubator (maintenance medium, MM), as previously described [35]. Cells were grown to 90% confluence in T-12.5 Falcon™ Tissue Culture Treated Flasks (Fisher Scientific, Waltham, MA, USA) in MM. For osteogenic induction, the cells were cultured in osteogenic media comprised of the above complete α-MEM medium supplemented with 10 mM β-glycerophosphate and 10 µg/mL ascorbic acid (differentiation medium, DM) and any experimental nutraceuticals. In preliminary studies, we optimized the initial seeding density to yield a robust monolayer of approximately 100k cells per flask at the time of collection (day 6 experimental). The media was refreshed by aspiration of 50% existing media and application of fresh media on the following schedule: 2 days, 2 days, and 3 days, thus generating a weekly cycle. Short-term experiments were collected after 6 days. Long-term experiments ended after 21 days.