Sodium pyruvate

The isolation of MSC followed a standardized protocol based on Ficoll (Ficoll Paque™ Plus, density 1.078 g/mL, GE Healthcare, Freiburg, Germany) density gradient centrifugation as reported previously [20 (link)]. 10 ×  10⁶ mononucleated cells (MNC) of each probe were cultivated in T75 tissue flasks in low-glucose DMEM (Gibco, Life Technologies, Darmstadt, Germany) culture media containing 10% (v/v) fetal calf serum (FCS; Biochrome, Berlin, Germany), 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM-glutamax, and 1 mM sodium pyruvate (all from Sigma-Aldrich).
Following the International Society for Cellular Therapy’s (ISCT) minimal criteria to define mesenchymal stromal cells (MSCs), we choose plastic adherence, as well as appropriate surface marker expression and trilineage differentiation for MSC characterization [21 (link)–25 (link)].

Adult male female Wistar rats were anaesthetised and sacrificed. The sciatic nerve was removed on both sides and transferred to a sterile work bench. The removal of epineurium and connective tissue was performed on ice. The nerves were kept in Hanks BSS (HBSS) + 1% (PAA Cölbe, Germany) Penicillin/Streptomycin (PAA, Cölbe, Germany) during preparation. Nerve fascicles were easily removed by pulling them out with sterile forceps. The fascicles were then cut into 3–4 mm pieces and transferred in 6-well dish (TPP, Trasadingen, Switzerland), followed by an incubation at 37 °C and 5% CO₂ with DMEM high glucose (Biochrome, Berlin, Germany), + 10% FCS (Biochrome, Berlin, Germany), + 1% P/S (PAA, Cölbe, Germany) and + 1% sodium pyruvate (Biochrome, Berlin, Germany) for 3 weeks. After 7 days, first migration of cells was observable [41 (link)].