Fig. 1: Functional properties and viability of SH-SY5Y cells cultured in different 2D and 3D conditions at different days of differentiation.

A Schematic representation of diverse protocols used to promote differentiation in SH-SY5Y cells (described in the Materials and Methods section). The main electrophysiological parameters of a mature neuron were measured in cells at 10 DIV; they were: densities of sodium and potassium currents evoked by a standard voltage protocol (B, C, D), resting membrane potential (E), and induced action potential firing frequency (F, G, H). The same parameters were measured for the conditions 3D DMAP1 Mix and 3D DMAP2 Mix, which turned out to be promising for inducing SH-SY5Y functional differentiation. (I–M) Evaluation of the different electrophysiological parameters at 10, 20, 30, and 40 DIV. N Representative spontaneous activity recorded from a differentiated SH-SY5Y cell. The percentage of cells with spontaneous activity in the 3D DMAP2 Mix increased up to 37% at 40 DIV (O). P SH-SY5Y cells were plated on 24‐well plates in the Growth Factor Reduced (GFR) basement membrane matrix. After 24 h, cells were differentiated using the 3D DMAP1 Mix or 3D DMAP2 Mix protocols, or left undifferentiated (Control). The number of viable cells was determined at 4, 7, 17, 27, 37, and 47 days for 3D DMAP1 Mix and 3D DMAP2 mix cultures; and at 4, 7, and 17 days for control cultures. The numbers of total samples analyzed for each time point described above were: 3, 4, 13, 2, and 4, samples for the 3D DMAP1 Mix condition; 3, 4, 11, 3, 3, and 13 samples, for the 3D DMAP2 Mix condition samples; 4 samples for the control condition. Q SH-SY5Y cells were plated on 96-well plates and differentiated as described in the Materials and Methods section (3D DMAP2 Mix) or left undifferentiated (Control). Cell cytotoxicity was quantified after 10, 20, 30, and 40 days of differentiation or after 7 days in culture for the control sample. The numbers of samples analyzed for each time point in two independent experiments were: control (n = 3), 10 DIV (n = 4), 20 DIV (n = 4), 30 DIV (n = 5), and 40 DIV (n = 13). Further details are described in the Materials and Methods section. Statistical tests used to determine the significance of differences among the conditions were: One-way ANOVA followed by Tukey’s multiple comparisons test, for data in panels C, D, H, and Q; Multiple t-tests, in panels K, M, and P (3D DMAP1 vs 3D DMAP2); Chi-Square test, in panel O. Significance is indicated as *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001.