Dmem low glucose

Umbilical cord blood-derived sdMSCs were isolated from newborn foals as previously described [14 (link)]. Briefly, 1XRBC lysis buffer (1.5 M NH₄CL, 100 mM KHCO₄, 1 mM EDTA, pH 7.3; Sigma, Oakville, Canada) was added to cord blood in 3:1 ratio and mixed for 10 min at room temperature. Then the mixture was centrifuged for 10 min at 400 g. The supernatant was discarded, and the cell pellet was washed in PBS (Sigma) and centrifuged. The supernatant was discarded, and cells were resuspended in sdMSC isolation media consisting of DMEM low glucose (Lonza, Walkersville, MD), 10% fetal bovine serum (FBS) (Invitrogen, Burlington, Canada), 100 nM dexamethasone (Sigma), 2 mM l-glutamine (Sigma), and 100 U penicillin–streptomycin (Invitrogen) and seeded in culture flasks at density 1 × 10⁶ cells cm⁻², incubated at 38°C and 5% CO₂.
Media were changed every 24 h for the first 3 days in culture and every 3 days thereafter. After the formation of putative sdMSC colonies, the adherent cells were detached with 0.25% trypsin (Sigma) and plated again in sdMSC expansion media consisting of DMEM low glucose (Lonza), 10% FBS (Invitrogen), 2 mM l-glutamine (Sigma), and 100 U penicillin–streptomycin (Invitrogen). Cells were passaged at 70% confluency and seeded at 5000 cells/cm².

BM-hMSCs were isolated as previously described17 (link). For this study we have used mainly cells at passage 3. For proliferation, cells were cultured at 37 °C in a humidified incubator with 5% CO₂ in maintenance medium (MM), low-glucose DMEM (Dulbecco’s modified Eagle’s) supplemented with 10% FBS, 1% glutamine, 50 μg/ml penicillin-streptomycin and amphotericin B (Lonza Group Ltd.). To induce osteogenesis, BM-hMSCs were maintained in Osteogenic Differentiating Medium (ODM), α-MEM (Minimum Essential Medium) supplemented with 10% FBS, antibiotics and the osteogenic mixture containing 100nM dexamethasone, 5 mM β-glycerophosphate disodium and 50 mg/ml ascorbic acid (Sigma-Aldrich, S. Louis, MO, USA). Treatment lasted up to 27 days and the medium was changed every 3 days. The study was conducted in accordance with the Review Board of Fondazione IRCCS Policlinico San Matteo and the University of Pavia (2011).