Developmental Steps for a Functional Three-Dimensional Cell Culture System for the Study of Asymmetrical Division of Neural Stem Cells

Stem cells are a cell type present during and following development, which possess selfrenewal properties, as well as the ability to differentiate into specific cells. Asymmetrical division is the cellular process that allows stem cells to produce one differentiated and one un-differentiated daughter cell during the same mitotic event. Insights in the molecular mechanisms of such process are minimal, due to the absence of effective methods for its targeted study. Currently, traditional methods of investigation include monolayer cell culture and animal models. The first poses structural limitations to the accurate representation of human tissue and cell structures, while animal models pose restrictions on the ability to visualize and manipulate the experiment performed, as well as being highly susceptible to error during data analysis and interpretation. Three-dimensional cell culture models are a novel approach to the study of cell mechanisms and disease, and are able to overcome many of the limitations of monolayer cell cultures and animal models. It is our goal to devise a three-dimensional cell culture system suitable for the in vitro study of asymmetrical division of neural stem cells at a single cell resolution. Current methods based on the culture of cells embedded in extracellular matrix-derived substrates have been efficient tools for the understanding of cellular mechanisms. However, currently available three-dimensional cell culture systems lack important qualities for an efficient application to the study of asymmetrical division of neural stem cells. A defining quality of cells of the neural lineage is the generation of spontaneous electrical activity, necessary for the transmission of signals in the organism. Current systems lack the ability to perform electrophysiological measurements and characterize the functionality of the cells in culture. A second limitation is posed by the adoption of non-specific substrates for cell culture able to recapitulate the natural cellular environment, as it is known that this has an impact on cell fate determination. Finally, current methods lack the ability to place and inject a controlled number of cells within the three-dimensional substrate, preventing the ability to perform studies at lower-cells resolutions. In this project, I developed three Aims with the goal of overcoming the three major limitations of three-dimensional cell culture systems. In Aim 1, I evaluated the efficiency of Microelectrode Arrays systems to perform electrophysiological measurements of neural stem cells and neuronal networks in vitro. In Aim 2, I fabricated and characterized a porcine brain extracellular matrix-derived hydrogel, and I tested its ability to promote stem cells survival and proliferation. Finally, in Aim 3, I optimized the parameters of a custom 3D extrusion-based bioprinter to perform single cell and single beads resolution printing. Combined, the development of these Aims allowed me to lay the foundations for the development of a functional threedimensional cell culture system applicable to the study of asymmetrical division in neural stem cells at a single cell resolution level, and which holds great potential for the uncovering of cellular mechanisms characteristic of stem cells and neurodegenerative disease.