Abstract:
Recently, the study of calcium (Ca²⁺) communication in the ‘Astrocytic Network’ has been suggested to be of equal importance to that of the traditional ‘Neural Network’. Astrocytes respond to external stimuli by increasing its cytosolic Ca²⁺ transiently, followed by the transmission of intercellular Ca²⁺ waves through the network. The objective of this thesis is to develop a neural engineered platform combining cell-patterning and laser cell-stimulation techniques that can interrogate an in vitro organised astrocyte network, thereby enabling the study of Ca²⁺ signals in organised in vitro astrocytic networks for the first time. In this thesis, we firstly investigate how to use parylene-C/SiO₂ substrates to design large grid astrocyte network in vitro. This is achieved by the design of grid networks with equally spaced nodes bridged by narrow lines. We demonstrate that astrocytes are isolated on nodes at the single-cell level and connected to neighbouring cells via narrow lines. Secondly, we extend this platform such that the astrocyte network can be interrogated by nanosecond laser stimulation. We determine the optimal laser power and inter-node distance of the network necessary for the successful Ca²⁺ signal propagation. Thirdly, we investigate the mechanism of Ca²⁺ initiation and propagation in the organised grid networks. We demonstrate that Ca²⁺ increases in the laser stimulated cells are mainly dependent on intracellular Ca²⁺ stores and the mechanisms of Ca²⁺ propagation to neighbouring cells are primarily mediated by extracellular ATP and a secondary smaller contribution via gap junctions. Finally, we demonstrate that hNT astrocytes respond to underlying geometric micro-shapes and preferentially extend cytoplasmic processes from the vertices of shapes. The organised networks combined with the laser stimulation developed in this thesis, provide a novel and robust platform for the study of the behaviour of networks of human astrocytes.