Development of brain-mimetic hydrogels for modelling neuronal differentiation

Biomechanically and biochemically tunable brain tissue models are notably essential for tissue engineering applications and neuroscience studies. Derivation of hydrogels through decellularization of native tissues is a promising strategy to reconstitute the native brain extracellular matrix for use in in vitro human models. Due to distinct features of the brain tissue and its implications on cellular behavior, it is particularly important to characterize and modulate the biochemical and biomechanical properties of constructed hydrogels from decellularized tissues. In the present study, we investigated the use of bovine brain tissue as a biomaterial carrier for neuroscience studies, assessed whether it could be an advantageous replacement for the human brain with easy accessibility, reproducibility and microenvironmental resemblance. We established and examined different methods for decellularization of bovine brain tissue and fabrication of reconstituted hydrogels. The decellularized tissues were evaluated with histological assessments and biochemical assays to both confirm elimination of cellular material and conservation of extracellular matrix components. Afterwards, decellularized tissues were solubilized with enzymatic digestion and reconstituted under physiological conditions in order to form hydrogels with thermal crosslinking capability. Mechanical characterization of hydrogels was performed to assess their stiffness and viscoelastic properties. Hydrogels were then tested for their three-dimensional cell encapsulation efficiency and their cytocompatibility with neuroblastoma cell line (SH-SY5Y) in culture. Collectively, it was shown that each decellularization technique resulted in different biochemical and biomechanical properties and these factors affected cell growth and behavior such as the degree of neurite formation. Given that mechanical microenvironment acts as an important parameter in cancer and neurodegenerative diseases, the results of this study provide significant insights. In the second part of the study, neuronal differentiation of neuroblastoma cells was investigated under 2D and 3D cell culture conditions to assess the effect of culture dimensionality and the presence of native brain matrix ligands on cellular fate. For this purpose, neuroblastoma cells were either grown on cell culture plate or encapsulated within decellularized brain-derived hydrogels. Following a neuronal differentiation regime, cells were evaluated morphologically through brightfield microscopy to determine neurite formation. Then, the expression of neuronal markers was assessed on both protein level by immunostainings and gene level by qRT-PCR. In conclusion, it was shown that synaptogenesis was improved by differentiated cells with elongated neurite formation in both 2D and 3D cultures. The proliferation rate was reduced and the gene expression levels of neuronal markers, including TUBB3 and CHAT were increased. Besides the common trends, significant differences were also observed between 2D and 3D cultured differentiated cells, whereas in 3D culture an increase in GFAP, glial cell marker, was detected.