293T cells, for AAV production, obtained from American Type Culture Collection (ATCC, Manassas, VA) were cultured in high-glucose Dulbecco’s modified eagle medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO), 100 U of penicillin/0.1 mg/ml streptomycin complex (Pen/Strep; Invitrogen, Carlsbad, CA). Primary stem-like GBM8 glioblastoma cells24 (link) were cultured as neurospheres in serum-free NeuroCult NS-A Basal Medium with Proliferation Supplement (StemCell Technologies, Vancouver, BC), supplemented with 20 ng/ml recombinant epidermal growth factor (EGF; R&D Systems, Minneapolis, MN), 10 ng/ml basic fibroblast growth factor (bFGF; Peprotech, Rocky Hill, NJ), and 2 µg/ml heparin (Sigma). All cells were cultured at 37 °C under 5% CO2 humidified atmosphere.
AAV vectors were produced as previously described.54 (link) Briefly, triple transfection of 293T cells using calcium phosphate precipitation was used with AAV2 ITR-containing single-stranded transgene plasmid, mini-adenovirus helper plasmid pFD6, and pH22 (AAV2 capsid; for in vitro use) or pAR9 (AAV9 capsid; for in vivo use) rep/cap plasmid. Three days later, the cells were harvested, lysed by freeze/thaw, and AAV was purified using iodixanol density gradient ultracentrifugation, resulting in typical yields of 1013 genome copies per ml (g.c./ml).
AAV vectors were produced as previously described.54 (link) Briefly, triple transfection of 293T cells using calcium phosphate precipitation was used with AAV2 ITR-containing single-stranded transgene plasmid, mini-adenovirus helper plasmid pFD6, and pH22 (AAV2 capsid; for in vitro use) or pAR9 (AAV9 capsid; for in vivo use) rep/cap plasmid. Three days later, the cells were harvested, lysed by freeze/thaw, and AAV was purified using iodixanol density gradient ultracentrifugation, resulting in typical yields of 1013 genome copies per ml (g.c./ml).