Dubecco s modified eagle s media dmem

Cos-7 and HEK293T cells (originally from ATCC) were cultured in Dubecco's modified Eagle's media (DMEM) from Sigma with 10% fetal bovine serum. Primary HUVECs (Lifeline Cell Technology, lot #1023) were cultured in human endothelial cell culture media, VascuLife EnGS (Lifeline Cell Technology, #LL-0002) (basal EnGS media, 0.2% EnGS, 5 ng ml⁻¹ rh EGF, 50 μg ml⁻¹ ascorbic acid, 10 mM L-glutamine, 1.0 μg ml⁻¹ hydrocortisone hemisuccinate, 0.75 U ml⁻¹ heparin sulfate and 2% FBS).

Human bone marrow was removed from the proximal femur during hip‐replacement surgery after approval from the North West Research Ethics Committee and with fully informed written consent of patients (male, n = 3; average age, 52 years (age range, 48 to 54 years)). hBM‐MSCs were isolated and expanded in Minimum Essential Media α (α‐MEM) (Sigma‐Aldrich, UK) with 20% FCS, as previously described.19 After 5 days, nonadherent cells were discarded, and adherent cells were cultured to confluence. hBM‐MSCs were subsequently cultured in α‐MEM supplemented with 10% FCS, antibiotics (100 U/mL Penicillin, 100 mg/mL streptomycin), 1% Glutamax and 10 uM ascorbic acid (referred to as hBM‐MSCs CCM). hBM‐MSCs at passage 3 were used for subsequent experiments.
Human osteosarcoma derived osteoblast cells (SaOS‐2) were purchased from Sigma (Sigma‐Aldrich, UK) and cultured in T75 flasks with Dubecco's modified Eagles media (DMEM) (Sigma‐Aldrich, UK) supplemented with 10% fetal calf serum (FCS), antibiotics (100 U/mL Penicillin, 100 mg/mL streptomycin) and 50 uM ascorbic acid (referred to as SaOS‐2 CCM). They were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂.