*2.2. 3D Model Preparation*

To obtain the scaffolds for 3D cultures of SH-SY5Y cells, sterile heterologous native lyophilized collagen type I sponge (BIOPAD™, Angelini Pharma Inc., Gaithersburg, USA) was cut using a sterile scalpel into pieces with squared dimensions able to fit 96-multiwell culture plates. Each piece was divided by subjecting it to a second longitudinal cut, performed in order to present a similar top surface as the cells. The pieces with approximately 1 cm<sup>2</sup> of surface area were inserted into a 24-multiwell plate and constituted the scaffolds for the cell culture. To establish the 3D SH-SY5Y culture, 50 μL of cell suspension in DMEM with 10% FBS was seeded atop of each scaffold. Different cell numbers per scaffold (50 × 103–100 × 103–200 × 103) were seeded to compare cell viability along the culture (1–6 days) and optimize cell seeding for differentiation. To differentiate 3D SH-SY5Y culture, a concentration of 4 × 10<sup>6</sup> cells/mL equivalent to 200 × 10<sup>3</sup> cells in 50 uL was used. After 45 min of incubation at 37 ◦C, 5% CO2, DMEM with 1% FBS, and 10 μM RA were added to the 3D culture in order to induce cell differentiation. The medium was changed every two days.
