Cell patterning and neuronal network engineering on parylene-C:SiO2 substrates
Hughes, Mark Antony
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Cell patterning platforms support diverse research goals including tissue engineering, the study of cell physiology, and the development of biosensors. Patterning and interfacing with neurons is a particular challenge, being approached via various bioengineering approaches. Such constructs, when optimised, can inform our understanding of neuronal computation and learning, and ultimately aid the development of intelligent neuroprostheses. A fundamental pre-requisite is the ability to dictate the spatial organization and topography of patterned neuronal cells. This thesis details efforts to pattern neurons using photolithographically defined arrays of the polymer parylene-C, printed upon oxidised silicon wafers. Initial work focused on exploring the parylene-C:SiO2 construct as a wide-ranging cell-patterning platform, assessing cell adhesion from both substrate- and cell-centric perspectives. Next, the LUHMES (Lund Human Mesencephalic) cell line was used to explore the potential for construction of interrogatable, topographically-defined neuronal networks. In isolation, LUHMES neurons failed to pattern and did not show any morphological signs of cellular differentiation. However, in the context of a cellular template (the HEK 293 cell line which was found to pattern reliably), LUHMES were able to adhere secondarily on-chip. This co-culture environment promoted morphological differentiation of neurons. As such, HEK 293 cells fulfilled a role analogous to glia, dictating neuronal cell adhesion and generating an environment conducive to neuronal survival. Neurites extended between islands of adherent cell somata. The geometry and configuration of parylene-C influenced the organisation of neurites. With appropriate designs, orthogonal neuronal networks could be created. The dominant guidance cue for neurite growth direction appears to be a diffusible chemotactic agent. HEK 293 cells were later replaced with slower growing human glioma-derived precursors, extracted during tumour debulking surgery. These primary cells patterned accurately on parylene-C and provided a similarly effective, and longer lasting, scaffold for neuronal adhesion.