Parylene/SiO2 Cell Patterning and Nanosecond Laser Stimulation for the Study of Calcium Signalling in Human hNT Astrocytes

Abstract

Astrocytes respond to external stimuli with intracellular calcium signals that can propagate through organized functional networks. Contemporary literature calls for the development of techniques that enable the study of astrocyte calcium signals in such organised networks. Existing techniques to culture astrocytes in vitro offer limited ability to control the interactions between cells. Similarly, existing techniques to stimulate astrocytes are difficult to restrict to individual cells and lack the spatiotemporal agility that is necessary for multiple precise stimulations. The objective of this work is to develop cell-patterning and cell-stimulation techniques that can physically manipulate astrocytic interactions in vitro, thereby enabling the study of calcium signals in in vitro astrocytic networks. In this thesis, we investigate how the parylene-C/SiO2 cell-patterning platform can be used to pattern astrocytes in vitro. We demonstrate how changing the underlying geometric pattern of parylene-C enables the isolation of single astrocytes and small multi-cellular astrocytic clusters. We then describe how cell patterning on the parylene/SiO2 platform can be significantly improved by selectively bonding polyethylene glycol to SiO2 areas. Additionally, this eliminates the previous requirement to use animal serum to activate parylene-C/SiO2 substrates. Next, we investigate calcium signalling in patterned astrocyte cultures. We demonstrate small isolated clusters of astrocytes replicate spatio-temporal clustering of calcium signalling that is observed in vivo but not in typical in vitro cultures. Finally, we demonstrate a new technique that uses a nanosecond UV laser to stimulate calcium signals in a fast and highly localized manner. Combined, cell-patterning and laser stimulation techniques will enable study the behaviour of astrocytes in a manner that more closely replicates the network like behaviour observed in vivo.