Preparation and Culturing of Primary Human Astrocytes and Glioma Cell Lines

Primary human cortical astrocytes were prepared from fetal tissue provided by Advanced Bioscience Resources (Alameda, CA) as described previously.⁴² Astrocytes were initially cultured in DMEM supplemented with 10% fetal bovine serum, non‐essential amino acids, pen/strep, sodium pyruvate and glutamine. Astrocytes were serum starved for the experiments. The KMWT1 cell line is a mouse glioma established from the spontaneous glioma tumor. We have used CRE‐inducible oncogenic lentiviruses to generate it. These viruses expressing p53 shRNA and shRNA to NF1 induce tumors in Gfap‐Cre mice with histopathology and molecular signatures similar to human GBM. Tumors are induced using oncogenic lentiviruses in B6.Cg‐Tg(Gfap‐cre)77.6Mvs/2J driver mice that express the mouse Gfap promoter‐driven CRE in astrocytes and a subset of adult neural progenitor cells (inducing their differentiation to oligodendrocyte progenitor cells and their expansion). Spontaneous gliomas are generated within 6–9 weeks with 86% efficiency (n = 39) (Mockenhaupt et al, in revision). KMWT1 and U251‐MG cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose (Corning) supplemented with 10% tetracycline‐free fetal bovine serum (Biowest). The HeLa cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) with 1.0 g/L D‐glucose (Lonza) supplemented with 2 mM L‐glutamine (Sigma‐Aldrich) and 10% fetal bovine serum (Biowest). Cell lines modified with the Sleeping Beauty transposon system were additionally supplemented with 1 μg/mL puromycin (Invitrogen). Cell lines were maintained at 37°C in a humidified atmosphere with 5% CO₂. Cells were examined regularly for mycoplasma contamination using PCR.⁴³