Culturing and Differentiation of SH-SY5Y Cells

SH-SY5Y cells (Sigma-Aldrich), a neuroblastoma cell line, were cultured in T75 flasks in standard culture medium containing DMEM and Ham’s F12 medium in a 1:1 ratio (VWR), supplemented with 10% fetal bovine serum (FBS; lot no. 11113, Bovogen, Keilor East, VIC, Australia) and 1% penicillin/streptomycin (PS; Westburg) in an incubator at 37°C and 5% CO₂. Cells were detached from the T75 flask using trypsin when reaching 70–80% confluency. Cells were then seeded at 1500 cells cm^-2 at 0 DIV onto the fibronectin-coated substrates in Petri dishes in standard culture medium. All cell cultures were seeded with cells at passage 19. As the PDMS substrate did not cover the complete Petri dish, cells on the fibronectin-coated Petri dish (polystyrene) surface were used as an additional flat surface control. Cells were left to adhere to the substrates for 3 h, after which the medium was replaced with culture medium supplemented with 10 μM retinoic acid (RA; Sigma-Aldrich) for 72 h to initiate differentiation of the cells and inhibit proliferation (Dwane et al., 2013; Teppola et al., 2016). Within the 72 h, the medium supplemented with RA was refreshed after 36 h. Subsequently, at 3 DIV, the medium was replaced with culture medium supplemented with 50 ng ml^-1 brain-derived neurotrophic factor (BDNF; Sigma-Aldrich) for 24 h to enhance neuronal differentiation (Encinas et al., 2000; Teppola et al., 2016). Cells were stored in standard culture medium until 21 DIV, with medium being refreshed every 48 h. Cultures were fixed after 21 DIV by washing the samples twice in PBS and subsequently treating them twice with 3.7% paraformaldehyde (Merck Millipore, Amsterdam, Netherlands) for 30 min.