Effect of the extracellular environment on astrocyte phenotype

Smith, Z H (2020) Effect of the extracellular environment on astrocyte phenotype. PhD thesis, University of Nottingham.

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Abstract

Astrocytes are a functionally and morphologically heterogeneous glial subtype essential for the development, function and survival of neurons in the healthy brain. Neuron-glial communication is therefore essential to normal brain function, but the mechanisms regulating signalling and anatomical association between these two cell types are still under investigation. The objective of this project was to develop in vitro models, validate the use of 3D culture techniques such as hydrogels and establish their potential for enabling astrocyte and neuron co-culture, and determine the possibility of combining 3D cell culture techniques with compartmentalized microfluidics devices. An additional aim was to discover the feasibility of replacing primary tissue with induced human astrocytes derived from fibroblasts.

2D in vitro cell culture environments are known to generate astrocytes with marked morphological differences to in vivo cells. 3D in vitro culture methods better resemble the in vivo microenvironment of astrocytes, therefore I investigated the effect of 3D matrices on cortical astrocyte phenotype and found that in both 2D and 3D environments astrocytes exhibit a highly heterogeneous range of morphological parameters. Astrocytes cultured in 3D hydrogels exhibited morphology that more closely resembled the in vivo phenotype compared with cells cultured in 2D. Additionally, considering that astrocytes are highly sensitive to their microenvironment, I have studied the effect of cell culture media on astrocyte phenotype and found that morphology was also highly responsive to the composition of cell culture media, particularly with the presence of serum. Thus the careful selection of cell culture media is crucial to reproduce conditions similar to those in vivo.

Neuron-glial communication in ex vivo slices of rat cerebellum was investigated by analysing how neuronal stimulation affected both presynaptic vesicle distribution and synaptic coverage by glial processes, as a physiologically relevant model of neuron-glial communication. Next I worked with in vitro systems with the potential to offer a similar level of analysis but with increased control of the extracellular environment. Compartmentalized microfluidics devices were used for the study of astrocyte-axon interactions, and the influence of astrocytes on neuron growth and development. Calcein AM loading determined that it is possible to both culture neurons and astrocytes in compartmentalized microfluidics devices, and within the 3D matrigel matrix, which is a step towards developing a superior in vitro model for cellular co-culture. To further improve the translational potential of this model, human induced astrocytes were derived from induced neural progenitor cells and morphological characterisations were conducted in 2D and 3D to determine if they are a suitable for studying human astrocyte complexity.

The ultimate aim of this PhD has been to investigate replacing classical 2D cell culture methods with 3D techniques, and replacing animal tissue with human tissue in order to provide physiologically relevant cell culture models which will facilitate greater understanding of astrocyte and neuron complexity, and their interactions in the brain. These data suggest that 3D hyaluronic acid hydrogels are a suitable culture system to replace 2D culture techniques with the potential to enable more translatable research into astrocyte physiology.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Bellamy, T C
Dajas-Bailador, F
Hathway, G J
Keywords: Astrocyte physiology,
Subjects: Q Science > QH Natural history. Biology > QH573 Cytology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 60487
Depositing User: Smith, Zoe
Date Deposited: 31 Jul 2020 04:40
Last Modified: 31 Jul 2020 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/60487

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