**3. Results**

#### *3.1. Development and Characterization of the 3D SH-SY5Y Culture System*

To fully assess biocompatibility between the collagen scaffold and SH-SY5Y cells, we evaluated cellular retention to the scaffold and cellular metabolism during 3D culture. The ability of SH-SY5Y cells to attach to the scaffold was evaluated by an MTT viability assay (Figure 2a). This assay was chosen because cells convert MTT to blue formazan that is retained by the cells and, for this reason, it makes it possible to distinguish cells attached to the scaffold from those released from the scaffold (see Figure S1). As reported in Figure 2a, about 95% of the total viable cells were able to attach after the switch from 2D to 3D culture conditions, while only 5% of the cells grew outside of the scaffold. To check the proliferation of SH-SY5Y cells, we used a Prestoblue assay as it makes it possible to monitor the metabolic activity of the same cell culture over time (Figure 2b). Cells were seeded at different concentrations, and cell viability was evaluated after 1 and 6 days. As expected, cell viability increased with an increasing numbers of cells per scaffold at both time points. Interestingly, the metabolic activity of the 3D culture after 6 days was comparable to that measured after 1 day at all tested seeding densities. Because the scaffold did not allow us to observe the cells under a microscope during growth, to verify that RA-treated SH-SY5Y cells were able to differentiate, we evaluated the mRNA level of the mature neural protein marker MAP2 as well as the secretable neurotrophin BDNF in 3D RA-treated cells (Figure 2c). Interestingly, both markers were upregulated in 3D RA-treated cells in respect to the 3D RA-untreated control, showing their ability to differentiate under 3D culture conditions. Figure S4 reports the macroscopic and microscopic appearance of the 3D SH-SY5Y culture.

**Figure 2.** Characterization of the 3D SH-SY5Y model. (**a**) Cellular adhesion to the scaffold was evaluated 24 h after cell seeding by MTT assay as reported in Materials and Methods. Data are expressed as a percentage of total viable cells and represent the mean of three independent experiments. (**b**) Metabolic activity of the 3D model was evaluated after 1 and 6 days from cell seeding by a Prestoblue assay as reported in Materials and Methods. Each bar represents the mean ± SEM of three independent experiments. Data were analyzed with a two-way ANOVA followed by the Fisher's test. \* *p* < 0.05. (**c**) Real time-PCR was performed in the 3D culture for neuronal markers. Each bar represents the mean ± SEM of three independent experiments, which were analyzed with an unpaired T-test. \* *p* < 0.05.

#### *3.2. SF, EGCG and PB Protect 3D SH-SY5Y Cells from Oxidative-Induced Injury*

Before studying the neuroprotective effect of SF, EGCG, and PB, we exposed 3D differentiated SH-SY5Y cells to 1 μM SF, 2.5 μM EGCG, 0.5 μM PB, or to a combination of the three compounds at the same concentrations (SEP) (Figure 3). These concentrations were chosen according to previous

reports where these concentrations were not very effective against oxidative stress [15–17]. Our results showed that all the tested concentrations—1 μM SF, 2.5 μM EGCG, and 0.5 μM PB—were not toxic.

**Figure 3.** Potential cytotoxicity of sulforaphane (SF), epigallocatechin gallate (EGCG), and plumbagin (PB) on SH-SY5Y cells. Cells were treated with 1 μM SF, 2.5, μM EGCG, and 0.5 μM PB, and after 24 h, viability was evaluated by a Prestoblue assay as reported in Materials and Methods. Results are expressed as a percentage of untreated cells. Each bar represents the mean ± SEM of three independent experiments, which were analyzed with a one-way ANOVA followed by the Fisher's test.

We then investigated the potential protective effect of the single treatments or a combination of them against H2O2-induced oxidative stress (Figure 4). As expected, incubation with 700 μM H2O2 for 2 h induced a significant reduction of cell viability compared to the control cells (Figure S2). Only 0.5 μM PB, 1 μM SF, and the co-treatment (1 μM SF, 2.5 μM EGCG, and 0.5 μM PB) were able to protect the cells against H2O2-induced damage. Of note, SEP co-treatment was the most effective treatment as it significantly increased cell viability compared to the other treatments.

**Figure 4.** Neuroprotective activity of SF, EGCG, and PB compounds against H2O2-induced damage. Cells were treated with 1 μM SF, 2.5 μM EGCG, and 0.5 μM PB, and after 24 h, were exposed to 700 μM H2O2 to induce oxidative stress. Cell viability in 3D cultures was measured by a Prestoblue assay as reported in Materials and Methods. Data are expressed as a percentage of untreated cells. Each bar represents mean ± SEM of three independent experiments. Data were analyzed with a one-way ANOVA followed by the Fisher's test. \* *p* < 0.05 vs. H2O2 treated cells; § *p* < 0.05 vs. sulforaphane, epigallocatechin gallate, and plumbagin (SEP) co-treatment.
