Streptomycin

For chondrogenic differentiation (MSC) or re-differentiation (AC), at the 3rd (MSC) or 2nd (AC) passages, 5 × 10⁵ of MSC or AC were resuspended in chondrogenic medium (DMEM high glucose, 0.1 μM dexamethasone, 0.17 mM ascorbic-acid 2-phosphate, 5 mg/ml transferrin, 5 ng/ml sodium selenite, 1 mM sodium pyruvate, 0.35 mM proline, 1.25 mg/ml bovine serum albumin (BSA), 100 I.U./mL penicillin, 100 μg/mL streptomycin, 5 mg/ml insulin (Sanofi-Aventis, Germany), and 10 ng/ml TGF-β1 (PeproTech, Germany)). Cells were centrifuged at 500 g for 5 min, or allowed to self-aggregate without centrifugation, to generate high-density pellets. Pellets were cultured for 3, 4, or 6 weeks at 37 °C, 6% CO² with medium change 3 times a week.

For chondrogenic differentiation, MSC were harvested after passage 4, and 5 × 10⁵ of cells were collected in 1.5-ml Eppendorf tubes by centrifugation (600g, 10 min), and subjected to high-density 3D culture in chondrogenic induction medium containing high-glucose DMEM supplemented with 0.1 μM dexamethasone, 0.17 mM ascorbic acid 2-phosphate, 5 μg/ml transferrin, 5 ng/ml selenous acid, 1 mM sodium pyruvate, 0.35 mM proline, 1.25 mg/ml BSA, 100 units/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin (Sanofi-Aventis, Frankfurt, Germany), and 10 ng/ml TGF-β1 (Peprotech, Hamburg, Germany). Pellets were cultured up to 6 weeks, with medium changed three times per week. To monitor chondrogenic differentiation, proteoglycan deposition was measured. For this, pellets were fixed with 4% paraformaldehyde, embedded in paraffin, and 5-μm sections were cut and stained with Safranin O solution (Safranin T Fluka Nr. 84,120, Fluka, Monte Carlo) and Fast Green (Chroma 1A 304, Chroma, Münster, Germany).

Primary cortical neuron cultures were prepared from the cerebral cortex of Wistar rat embryo of 17 days as described previously (Kim et al., 1998 (link)). Briefly, pregnant Wistar rats were anesthetized with sodium pentobarbital (30 mg/kg, i.p., Sigma–Aldrich) and sacrificed by cervical dislocation. The cerebral cortex of fetal rats was rapidly removed bilaterally and collected. Tissues were then gently minced using a sterile razor blade and digested in PBS (0.1 M, pH 7.4, Sigma–Aldrich) for 15 min. A Pasteur pipette was used for dissociation of cells (approximately 5–10 times). After centrifugation (200 × g for 3 min), cells were re-suspended in DMEM (Sigma–Aldrich) supplemented with FBS (15%, Carlsbad), L-glutamine (2 mM, Sigma–Aldrich), sodium bicarbonate (4.2 mM, Sigma–Aldrich), BSA (0.3 g/l, Sigma–Aldrich), β-mercaptoethanol (0.1 mM, Sigma–Aldrich), penicillin (1%, Sanofi Aventis), streptomycin (50 μg/ml, Sanofi Aventis) and grown on 0.1% poly-L-Lysine (Sigma–Aldrich) coated plates. Cultures were incubated at 37°C in a humidified 5% CO₂ atmosphere. To prevent proliferation of non-neuronal cells, cytosine β-D-arabinofuranoside hydrochloride (10 μM, Sigma–Aldrich) was added 3 days after plating. In all experiments, 11 days mature cells were used.