Purification and Propagation of Schwann Cells

Schwann cell cultures at passage 11 were taken from storage in liquid nitrogen and thawed as required. These cells were previously derived from primary neonatal Schwann cells (SCs) obtained from Wistar RjHan:WI rat pups and cultured and purified following a previously published protocol [48 (link)] and in accordance to [30 (link)]. In brief, sciatic nerves were collected and enzymatically digested. The separated cells were cultured in poly-l-lysine (PLL)-coated dishes containing Dulbecco’s modified Eagle’s medium supplemented with 0.1 µM Forskolin, 1% Pen/strep, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal calf serum (FCS) (all from Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO₂ in a humidified atmosphere. PLL coating was performed by covering the culture dishes with PLL for 45 min at room temperature. After removing the PLL, plates were washed 2 times with Ampuwa^® (Fresenius Kabi, Bad Homburg vor Höhe, Germany). To prevent excessive fibroblast contamination, 1 mM of arabinoside-c (Sigma-Aldrich, St. Louis, MO, USA) was added at 2 and 3 days in vitro, respectively. The cells were finally purified to >90% by immunopanning, purity was controlled in immunocytochemistry, and cells propagated in medium as described above, just with an increased concentration of 2 µM Forskolin [30 (link)].