Dmem high glucose

Normal Articular Chondrocytes (HACs) were isolated from ten young patients (aged 19–38 years) subjected to knee arthroscopy for traumatic lesions, as described in Caron et al. (22 (link)). Dedifferentiated cells were seeded at 10 × 10³ cells/cm² and cultured in DMEM High Glucose, 10% FBS, FGF-2 10 ng/ml and TGFβ₁10 ng/ml (R&D systems) in monolayer culture until confluence. After 1 passage, the cells were trypsinized from monolayer cultures, counted and divided in polystyrene 15 ml tubes. Each tube containing 5 × 10⁵ cells was centrifuged at 500 g for 10 min at 4°C. The resultant pellets were not resuspended, but cultured in chondrogenic medium for 28 days. The medium was replaced every 3 days and at the end of the culture half of the pellets from each group (Ctr and T-Lys) were collected for histological analysis and the remaining pellets from each group were used for RNA extraction.

Sciatic nerves were isolated from P0-P1 rats, and digested with 1 mg/ml collagenase A (Sigma). Schwann cells were plated into poly-D-lysine (Sigma) coated dish and grown in DMEM High Glucose (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1 mM Ara-C (Sigma) for 3 days. After passage, Schwann cells were cultured with proliferation medium (DMEM High Glucose supplemented with 10% fetal bovine serum, 10 ng/ml NRG1 (R&D Systems), 5 μM forskolin (Sigma), 1% L-glutamine (Hyclone, Cat# SH30034) and 1% penicillin/streptomycin). To differentiate, Schwann cells were cultured in differentiation medium (DMEM High Glucose supplemented with 0.5% fetal bovine serum and 1 mM dibutyl cyclic AMP (Sigma) with 1% L-glutamine and 1% penicillin/streptomycin).