3D Neuroblastoma Cell Culture Model

SK-N-BE(2), SH-SY5Y human NB cell lines were chosen from a variety of available cell lines, since MYCN-amplified and ALK-mutated tumors represent 64% of high risk neuroblastoma (50 and 14%, respectively). SK-N-BE(2), SH-SY5Y, and SW10 mouse Schwann cell lines were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). NB cells were expanded in supplemented IMDM medium (Gibco, Life Technologies, Waltham, MA, USA) and SW10 in supplemented DMEM (Gibco, Life Technologies, Waltham, MA, USA) at 37 °C and 5% CO2 atmosphere. Two dimensional cell cultures were grown in 8-well Cell Culture Slides (SPL Life Sciences, Waltham, MA, USA), until they reached 60% confluence before immunocytochemistry (ICC) analysis. Bioinks for 3D culture were formed by mixing SK-N-BE(2) or SH-SY5Y cells with the prepolymer solution at 37 °C to a final solution of 2 × 10⁶ mL⁻¹, as previously reported [21 (link)], including in some cases, an additional 2 × 10⁵ mL⁻¹ of SW10 cells. Hydrogels were cultured from 2 to 12 weeks in supplemented IMDM medium replaced every 2 or 3 days before immunohistochemistry (IHC) analysis.