Establishment and Maintenance of Medulloblastoma PDXs

Three group 3 medulloblastoma xenografts [21 (link), 22 ] established from pediatric patients were used for experiments: D341 Med (D341), D384 Med (D384), and D425 Med (D425); kindly provided by Darell D. Bigner, MD, PhD, Duke University Medical Center [23 (link), 24 (link)]. The xenografts were maintained through serial passage in athymic nude mice (Envigo, Pratville, AL). Tumors were harvested and cells were dissociated using a Tumor Dissociation Kit (Miltenyi Biotec, San Diego, CA) per manufacturer’s protocol. Cells were then washed in Roswell Park Memorial Institute (RPMI) 1640 medium, spun (150 × g × 6 minutes), and debris removed using a 70 μm cell strainer (Corning Inc., Corning, NY). The cells were maintained under standard culture conditions at 37°C and 5% CO₂, in neurobasal (NB) medium (Life Technologies, Carlsbad, CA) supplemented with B-27 supplement without Vitamin A (Life Technologies), N2 supplement (Life Technologies), amphotericin B (250 μg/mL), gentamicin (50 μg/mL), L-glutamine (2 mM), epidermal growth factor (10 ng/mL; Miltenyi Biotec), and fibroblast growth factor (10 ng/mL; Miltenyi Biotec). All three medulloblastoma human PDXs were verified within the last 12 months using short tandem repeat analysis (Heflin Center for Genomic Sciences, UAB, Birmingham, AL).