**1. Introduction**

Oxidative stress is strongly involved in the pathogenesis of different neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [1]. Particularly, an excess of reactive oxygen species (ROS) released by cells promotes oxidative stress, which is a cause of tissue injury and results in dysfunction in the nervous system. So far, no drugs have been discovered to prevent or halt the progression of these widely spread neurological disorders, and the treatments available only manage the symptoms. Therefore, there is an urgen<sup>t</sup> need for new treatments for these diseases, since the World Health Organization (WHO) predicts that by 2040, neurodegenerative diseases will become the second-most prevalent cause of death [2]. New therapeutic approaches can derive from phytochemicals, a huge source of compounds that have been widely investigated in the last years [3]. Sulforaphane (SF) (Figure 1a) is an isothiocyanate derived from *Brassicae* vegetables and has consistent support in the literature for its preventive role against oxidative stress [4], in addition to its well-known role in chemoprevention [5]. Epigallocatechin gallate (EGCG) (Figure 1b), the major catechin found in green tea [6] with diverse medical potential [7] has been demonstrated to promote neuroprotection in numerous studies [8]. Plumbagin (PB) (Figure 1c), a naphthoquinone isolated

from the *Plumbaginacae* family, has mainly been studied in respect to its anti-inflammatory [9–11] and antimicrobial activities [12], even though it was also shown to modulate oxidative stress response [13] and precisely target NADPH oxidase 4 (NOX4) [14]. Importantly, the treatment of undifferentiated SH-SY5Y cells with a specific concentration of SF [15], EGCG [16], and PB [17] has been reported to decrease oxidative stress at different levels. On this basis, we hypothesized that a proper combination of such bioactive compounds could possess a higher effect in counteracting oxidative stress-induced neurodegeneration. Within organs, there are various concentration gradients for oxygen as well as for effector molecules, i.e., internal metabolites or exogenous compounds/drugs. Since the human brain cannot be modelled adequately in animals [18], reductionist humanized cellular systems are used and increasingly requested according to the 3Rs (Replacement, Reduction and Refinement) rule.

**Figure 1.** Chemical structures of the natural compounds used in this study. (**a**) Sulforaphane (SF), (**b**) epigallocatechin gallate (EGCG), (**c**) plumbagin (PB).

Currently, the majority of cell-based studies on neurodegeneration have been carried out on cultured cells propagated in two dimensions on plastic surfaces. However, cells cultured in these non-physiological conditions do not represent the cells residing in the complex microenvironment of the brain. With respect to this, recent evidence has suggested that three-dimensional (3D) in vitro microenvironments may better model key features of brain tissues in order to study the molecular mechanisms at the base of neurodegeneration and neurorepair [19,20]. Numerous in vitro approaches have been carried out to mimic human neuronal features, based on neuronal-like cells such as the neuroblastoma line SH-SY5Y. SH-SY5Y is a human cell line that divides quickly and has the ability to differentiate in post-mitotic neurons, thus it is considered a convenient model to study Parkinson's [21] and Alzheimer's diseases [22]. Unlike traditional two-dimensional (2D) cultures, the different availability of oxygen and growth factors in a 3D cell culture should expectantly favor a more in vivo-like morphology and growth of these cells. Indeed, several 3D culture models have been developed with SH-SY5Y cells, in terms of cell aggregates [23], spheroids [24,25], or including different scaffolds [26–28]. To support 3D cultures of SH-SY5Y or neuronal cell lines, collagen has also been used, like collagen hydrogel [29] or porous collagen-based scaffolds [26,30]. To the best of our knowledge, none of the previous models has been used to investigate the ability of natural bioactive molecules to confer resistance to oxidative stress. The aim of this study was to evaluate the neuroprotective effect of a combination of SF, EGCG, and PB in preventing cell damage derived from oxidative stress in a 3D cell culture based on a collagen porous scaffold.
