The effect of culture dimensionality and brain extracellular matrix in neuronal differentiation

Abstract

Objective: Neuroblastoma cells are frequently used in neuroscience studies due to their human origin and ability of extensive propagation compared to animal-derived primary neuron cultures. Although they are tumor-derived, they exhibit neuronal differentiation capability in the presence of several agents including retinoid acid. Several studies have quested for successful differentiation protocols and faithful representation of neuronal characteristics. However, they predominantly pursued conventional two-dimensional (2D) cultures where the role of three-dimensional (3D) tissue microenvironment and cell-matrix interactions remained unknown. In this study, we investigated the effect of culture dimensionality and native brain extracellular matrix (ECM) on neuronal differentiation of neuroblastoma cells. Materials and Methods: Decellularized brain ECM hydrogels offer a physiologically relevant in vitro 3D culture platform with the representation of key biochemical and biophysical aspects of the native tissue microenvironment for modeling cellular processes. We cultured SH-SY5Y cells on 2D or as encapsulated in 3D decellularized brain ECM hydrogels and assessed them for morphological shift, neurite extension, and expression of neuronal, synaptic, astrocytic, cholinergic, stemness, proto-oncogene and neuropathological markers. Results: Our findings demonstrate that the 3D brain ECM microenvironment distinctly affects the differentiation process compared to conventional culturing. In 3D ECM, neuronal differentiation occurred as in 2D, with upregulation of neuronal markers, change in cell morphology, and promotion of neurite extension. However, during differentiation, maintenance of stemness was observed in a 3D-specific manner. Furthermore, 3D differentiation promoted significant upregulation of astrocytic and synaptic markers which was not observed in 2D. Conclusion: This study highlights the importance of physio-mimetic 3D brain models.