3D Matrigel Sandwich Culture of Neuroblastoma Cells

3D multicellular spheroids were cultured based on a previous study [9 (link)]. A modified protocol was developed to form the Matrigel sandwich structure for 3D in vitro models, based on the previous reports from our lab [11 (link)] as shown in Fig. 1. Briefly, to form the bottom layer of Matrigel, 50 µL Matrigel (Sigma-Aldrich) was added into each well which had been pre-chilled on ice, and then the plates were incubated at 37 °C for 30 min, allowing the Matrigel to polymerise. SH-SY5Y cells in 10% Matrigel (v/v) in 500 µL pre-chilled EBM-2 supplemented with 2% FBS and 1% penicillin–streptomycin were added onto the polymerised Matrigel at the density of 3–5 × 10⁴ cells/mL. In the co-culture model, HUVECs were added together with SH-SY5Y onto the polymerised gel layer at the density of 1–1.5 × 10⁵ cells/mL in 500 µL endothelial basic medium (EBM-2, Lonza, UK) supplemented with 2% FBS (Life Technologies, US) and 1% penicillin–streptomycin (Thermo Fisher). The 3D in vitro systems were maintained at 37 °C, 5% CO₂ for at least 24 h before further drug testing.Fig. 1

Schematic procedures for 2D and 3D in vitro models used in this study. a 2D monolayer culture of SH-SY5Y; b 3D Matrigel sandwich model of SH-SY5Y. c 3D Matrigel sandwich co-cultured with vascular endothelial cells HUVECs model of SH-SY5Y