Developmental Steps for a Functional Three-Dimensional Cell Culture System for the Study of Asymmetrical Division of Neural Stem Cells
Creators: name: Zamponi, Martina
Publisher: name: Old Dominion University Libraries
Year: 2018

description: Stem cells are a cell type present during and following development, which possess self-
renewal 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 three-
dimensional 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.