Fig. 3.

Neuron-like cells cultured in 3D retain a dopaminergic phenotype. (A) Cell number optimisation of 3D-differentiated SH-SY5Y using LDH cell viability assay. An incremental increase in cell density is associated with decreased cell viability (∼17% decrease from 1×10⁶ cells/ml to 1.25×10⁷ cells/ml). LDH cell viability assay was normalised to cell-density-matched positive controls. All data points are present, with mean shown as a straight line of at least four independent repeats. (B) mRNA levels of genes specifically associated with dopaminergic neurons were evaluated by qPCR in different cell densities. Relative gene expression normalised to reference genes (ACTB and YWHAZ) and undifferentiated SH-SY5Y. Expression of neuronal (TUBB3, MAP2 and NES) and dopaminergic (DAT, DRD2 and VMAT2) markers was evaluated in cell densities. A density of 7.5×10⁶ cells/ml demonstrated increased expression of DRD2, MAP2 and VMAT2, and was chosen for further experiments. Data presented as mean±s.e.m. of at least three independent experiments. (C) 3D-differentiated SH-SY5Y cultures retain a post-mitotic neuronal dopaminergic phenotype in 3D as determined by the absence of Ki67 staining and positive staining for NF200 and dopamine. Positive control for Ki67 staining is undifferentiated SH-SY5Y cultured in 3D. Scale bars: 100 µm.