Establishing Neuroblastoma PDX Cell Cultures

Tissue from neuroblastoma PDXs was dissociated and digested for 45 min at 37 °C with Liberase (0.15 mg/mL, Roche), passed through a 70 µm cell strainer (BD Biosciences) and cultured in stem cell medium (SC medium) at 37 °C in 5% CO₂^{10 (link)}. Spheres were dissociated using Accutase (Sigma-Aldrich, St. Louis, MO). PDX #2 had been established from cerebral metastasis from a stage 4 tumor where the patient had undergone prior treatment while PDX #3 had been established from a primary stage 3 tumor in the adrenal gland. For serum culture, SC medium excluding growth factors and B-27 supplement was supplemented with fetal bovine serum (FBS) or 10% charcoal-stripped FBS (Thermo Scientific). For monolayer culture on human recombinant laminin (Biolamina), plates were coated according to manufacturers instructions. PDX cells were routinely grown to confluence and dissociated using Accutase. The neuroblastoma cell line SK-N-BE(2)c (ATCC) was cultured as previously described^{38 (link)}. All cells were routinely screened for mycoplasma and authentication was performed by SNP profiling of SK-N-BE(2)c cells, PDX cells and corresponding PDX (Multiplexion, Germany).
Treatment with MYC-MAX inhibitor 10058-F4 (Sigma) was performed for 72 h at 60 μM or 75 μM. Treatment with neurotrophins was done for 4 days with nerve growth factor (NGF, 50 ng/ml, PeproTech), neurotrophin-3 (NT-3, 50 ng/ml, PeproTech), or a combination. Prior to neurotrophin treatment, cells were cultured in SC medium, 2% or 10% serum for 7 days. For cytostatic treatments, cells were treated with cisplatin, doxorubicin or etoposide in BRANDplates® 96-well plates (VWR) directly after seeding or 48 h post-seeding. CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to determine cell viability 72 h post-treatment. Cell culture images were captured with a Carl Zeiss AxioCam IC microscope and the ZEN software.