3D Bioprinted differentiated murine neurospheres as a neurogenesis model

Abstract

AbstractBackgroundAdult neurogenesis, the generation of neurons by neural stem cells (NSCs), occurs mainly in neurogenic niches, located in the dentate gyrus of the hippocampus and in the subventricular zone (SVZ) of the lateral ventricles. However, in Alzheimer’s disease (AD), proliferative activity of NSCs and the generation of new neurons is deregulated. Although there are animal models of AD, developing an in vitro three‐dimensional (3D) model of the disease may certainly enhance the approach of a multidinamic microenvironment able to evaluate specific areas of pathogenesis. The aim of this study was to produce 3D constructs targeting their application as an in vitro model of neurogenesis.MethodNeurosphere (clusters of NSCs and neural progenitor cells) cultures were developed from isolated cells from the SVZ of 6 weeks old C57bl/6 mice. Cells were cultivated in poly‐HEMA coated flasks for 7 days, then, formed neurospheres (∼100µm) were dissociated and bioprinted in an alginate:gelatin solution (6%:4%, respectively). The bioink was 3D printed, crosslinked with 2% CaCl2, and the constructs were maintained for 2 days in culture medium to allow neurosphere expansion. From days 3‐7, the constructs were cultivated in growth or differentiation medium, to stimulate neuronal differentiation. Morphological organization of cells within the neurospheres was evaluated.ResultThe produced 3D constructs are cytocompatible and allow neurosphere formation and growth under growth condition (Fig. 1A). As the growth medium was substituted by the neuronal differentiation medium, processes outgrowth was observed spreading outside the spheres (Fig. 1B).ConclusionA biocompatible construct loaded with neurospheres was produced by 3D bioprinting. The outgrowth of cellular processes outside the spheres suggest that neuronal differentiation occurs, showing that the biomaterial consists in an excellent model to investigate neurogenesis and neurogenic niches dynamics, two impaired elements in AD pathogenesis. Further steps of this study include the improvement of the 3D constructs, creating a microenvironment suitable to better simulate the potential of cellular differentiation under AD pathological conditions. Acknowledgments: This study was supported by São Paulo Research Foundation (FAPESP, grants 2022/08664‐4 and 2018/45605‐8), and by the Brazilian National Council for Scientific and Technological Development (CNPq, grant 406258/2022‐8).