Dispase grade 1

Ethical approval is granted and patients gave consent with the usage of the tumour samples to isolate of human primary schwannoma cells. The methods were described by Rosenbaum et al.^{58 (link)} Briefly, schwannomas were surgically removed under local anaesthesia, and were then preincubated for 1–7 days in incubation medium (DMEM plus 10% FBS, 500 U/ml penicillin/streptomycin, 0.5 μM Forskolin, 2.5 μg/ml Amphotericin B) in 10% CO₂ and then dissected into 1-mm-long pieces in DMEM with 10% FCS containing 500 U/ml penicillin/streptomycin, 160 U/ml collagenase type I (Sigma) and 1.25 U/ml dispase grade I (Roche, West Sussex, UK). Tissue pieces were incubated in proteolytic enzymes for 24 h before they were dissociated by trituration with a narrowed Pasteur pipette. Cell suspension was added to a 50-ml Falcon tube. Cells were collected and resuspended in proliferation medium: DMEM with 10% FCS, 500 U/ml Pen/Strep, 0.5 μM Forskolin (Tocris, Abingdon, UK), 2.5 μg/ml Amphotericin B, 10 nM b1-heregulin (R&D System, Abingdon, UK) and 2.5 μg/ml insulin (Sigma). Cells were seeded into 96-well plates (Greiner Bio-one, Stonehouse, UK), coated with 1 mg/ml poly-l-lysine (Sigma) and 4 μg/ml natural mouse laminin (Life Technologies, Paisley, UK), at a density of 3000 cells/well. Proliferation medium was changed every 3–4 days and cells were passaged when confluent.