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Durham e-Theses
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Investigating the impact of the 2D mechanical environment on skin cells cultured in vitro

HUNTER-FEATHERSTONE, EVE,FLOYD (2021) Investigating the impact of the 2D mechanical environment on skin cells cultured in vitro. Doctoral thesis, Durham University.

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Abstract

Mechanotransduction describes a cell’s ability to sense mechanical stimuli imparted by its surroundings and translate them into biochemical signals. These signals subsequently influence cell behaviour by promoting remodelling of the cytoskeleton, changes to gene expression and tumour suppression. This is particularly important for tissues with a high cell turnover like the skin. Fibroblasts and keratinocytes, the skin’s predominant cell populations, have been shown to respond to physical stimuli such as stretch, compression and shear forces, leading to changes in collagen deposition, and proliferation and migration.

During in vitro cell culture it is conventional to use extremely stiff substrates that do not reflect the physiological microenvironment (e.g., plastic and glass). Whilst there have been attempts to limit this issue with the development of 3D tissue models, cells are still first being cultured, or “primed”, on a highly stiff surface.

The aim of this project was to investigate keratinocyte and fibroblast behaviour, phenotype, and genotype expression when cultured on biomimetic hydrogel-coated dishes with similar mechanical properties to their in vivo environment. It was hypothesised that optimisation of this 2D culture environment would facilitate the development of 3D skin equivalents that better reflected in vivo tissue than current models grown from cells primed on plastic.

This study showed that primary keratinocytes and fibroblasts cultured on biomimetic substrates were more reminiscent of cells in quiescent skin rather than the activated phenotype observed on plastic. Keratinocytes exhibited reduced nuclear and cytoplasmic stiffness in response to a 4 kPa culture substrate, and changes were observed in the expression of proliferation and differentiation markers, and proteins involved in mechanosensing. Epidermal 3D models produced using keratinocytes primed on 50 kPa dishes were thicker and better organised. Fibroblasts cultured on soft 2D substrates of 1 kPa and 4 kPa were observed to have fewer reactive oxygen species and expressed fewer DNA damage markers following irradiation to induce senescence.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Faculty and Department:Faculty of Science > Biological and Biomedical Sciences, School of
Thesis Date:2021
Copyright:Copyright of this thesis is held by the author
Deposited On:23 May 2022 13:55

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