Preparation of adult NPC. Rats were deeply anesthetized using a cocktail of ketamine (62.5 mg/kg; WDT, Garbsen, Germany), xylazine (3.175 mg/kg; WDT, Garbsen, Germany) and acepromazine (0.625 mg/kg, Sanofi- Ceva, Düsseldorf, Germany) in 0.9% ster- ile saline solution and killed by decapita- tion. The region of the complete cervical enlargement (spinal cord level C3 through T1) was dissected out. After removal of the dura, the tissue was minced, washed in sterile Dulbecco’s phosphate buffered saline/D-glucose (4.5 g/l; PAA Labora- tories, Linz, Austria) and digested in a solution of papain (0.01%; Xxxxxxxxxxx Biochemicals, Lakewood, USA), neu- tral protease (0.1%; Roche, Mannheim, Germany), DNase I (0.01%; Xxxxxxxxxxx Biochemicals) and 12.4 mM MgSO4, dis- solved in Hank’s balanced salt solution (HBSS; PAA Laboratories, Linz, Austria) for 30 min at 37°C. The digested tissue was centrifuged at 120 x g for 5 min at 4°C and washed three times in DMEM-HAMS F12 (Pan Biotech, Aidenbach, Germany), supplemented with 10% fetal calf serum (FCS; Pan Biotech, Aidenbach, Germany). The cells were transferred to culture dish- es containing serum-free growth medium, which consists of Neurobasal medium with B27 supplement (both Gibco, Karl- sruhe, Germany) and 20 ng/ml recombi- nant human FGF-2 (R&D System, Wies- baden, Germany). Neurobasal medium with B27 supplement has been shown to substantially increase the proliferation rate of NPC in vitro 22 as compared to stan- dard proliferation medium consisting of DMEM/F12 and N2 supplement 23, 24. Cells were either grown as neurospheres in un- coated cell culture flasks or as adherent monolayers in culture flasks, which were coated as follows: flasks were incubated with 20 µg/cm2 poly-l-ornithine (Sigma) in distilled H2O for 2 h at 37°C, rinsed and incubated with 0.4µg/cm2 laminin (Sigma) in PBS for 2 h at 37°C. The cell culture medium was changed twice per week. In neurosphere cultures, the medium was replaced by centrifuging the medium con- taining neurospheres at 120 x g for 5min at 4°C, removing the supernatant and re- suspending the cells in fresh growth me- dium. Cell cultures were passaged in 2 week intervals. Monolayer cultures were detached by incubation with 40 ml/cm2 Acccutase (Innovative Cell Tech, San Diego, USA) for 5min at 37°C. Finally, the cells were centrifuged at 120 x g for 5min at 4°C and resuspended in fresh growth medium. Passaging of neurospheres was performed as follows: the medium con- taining the neurospheres was collected in a ...
