Document Type

Theses, Ph.D

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Disciplines

1.3 PHYSICAL SCIENCES

Publication Details

Thesis successfully submitted to Technological University Dublin in fulfilment of the requirements for the award of Doctor of Philosophy (PhD)

Abstract

The aim of this work was to investigate changes to the molecular composition and conformation of HaCaT cells as a result of simulated solar radiation in a 3D in vitro skin model by Raman spectroscopy. The process to achieve this goal was performed in three main stages: (1) optimisation of the working concentration and volume of two 3D membranes, used as a structural support in the skin model; (2) the construction of the 3D in vitro skin model and; (3) the investigation of the dose-dependent effects of solar radiation on HaCaT cells in the skin model in comparison with the conventional 2D models. The novelty in the 3D culture models is that they have an increased physiological representation of in vivo-like conditions, compared to 2D cell culture models. The introduction of the extracellular matrix enables cells to achieve their natural morphology and polarity, and it improves the mechanical/biochemical signals and cell-microenvironment communication. Moreover, 3D cell cultures offer alternative to animal models, following the regulations against human and animal testing (EU Directive-2010/63/EU and US Public Law 106-545, 2010, 106th Congress). In the first stage, the results revealed that the cell geometry in 3D cultures modifies the uptake and conversion rates of the cytotoxicity assay dye in comparison with 2D models, resulting in an apparent increment in cell viability levels. However, flow cytometry showed no differences in live cells and apoptosis levels between 2D and 3D cultures, although a cell cycle arrest at the S-phase in a cancer cell line cultured in collagen I was observed. The results of this study promotes the use of collagen I and Geltrex in the construction of a 3D in vitro skin model, since the cellular health and viability levels are not affected by these extracellular matrices. The second stage in this iii thesis illustrates the methodology to build the skin model. Firstly, human dermal fibroblasts were embedded in collagen I to form the dermis layer. Secondly, after 1 day of incubation, HaCaT cells cultured in a Geltrex layer were seeded on top of the dermis layer to form the epidermis in the in 3D vitro model. The ensemble of these two layers resulted in a simplistic 3D in vitro skin model. In the optimisation of the model, the use of human serum to supplement the media for the cell culture was seen to affect the viability levels of cells in both 2D and 3D models. Thus, the traditional foetal bovine serum was employed in the cell culture. In the final stage, the influence of using 3D matrices in HaCaT cells exposed to simulated solar radiation in comparison with 2D models is reported. The detrimental effects of solar radiation on cell integrity were studied using different techniques. The induced morphological changes were observed through histochemical staining in 2D models as well as the characterisation of the 3D skin model. The viability levels in both culture systems (2D and 3D) were monitored using the colorimetric assay Alamar blue. The viability results suggested that solar radiation had no effects on cell health immediately after irradiation. However, this was associated with the performance of the Alamar blue dye in the 3D membrane. The investigation of the photobiological events occurring at the molecular level in the cell due to the impact of simulated solar radiation was performed by Raman microspectroscopy. The focus was on the cell nuclei, as DNA is the main target of solar radiation. As an immediate effect of simulated solar radiation and cell interaction, Raman spectroscopy suggests induction of single strand breaks, formation of bipyrimidine photoproducts and oxidative damage of bases, whereas as a later-term response, protein damage is observed. Hence, the spectral analysis showed that not only cell cycle is affected when cells are transferred from 2D models to a more complex system as 3D models. Cell responses to external stimuli, drugs or compounds are altered in this transition.

DOI

https://doi.org/10.21427/94g0-ng44

Funder

Consejo Nacional de Ciencia y Tecnologia (CONACYT); Consejo Zacatecano de Ciencia, Tecnología e Innovación (COZCYT)

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