Penicillin

After expansion, MSC were subjected to adipogenic induction medium consisting of DMEM high glucose, 10% FCS, 1 μm dexamethasone, 0.2 mM indomethacine, 0.5 mM isobutylmethylexanthine (all Sigma, Deisenhofen, Germany), 0.01 mg/ml insulin (Sanofi-Aventis, Frankfurt, Germany), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured for 3 weeks with medium changed twice per week. To monitor adipogenesis, cells were fixed with 4% paraformaldehyde and stained with 0.3% Oil Red O solution (Chroma, Münster, Germany). Dye was re-extracted from vacuoles by 60% isopropanol and quantified by measuring its optical density at 490 nm.

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.