Developing a New Drug Delivery System for the Brain

Carruthers, Sam (2021) Developing a New Drug Delivery System for the Brain. MRes thesis, University of Lincoln.

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Developing a New Drug Delivery System for the Brain
MRes Thesis
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Carruthers, Sam - Life Science. December 2021.pdf - Whole Document

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Item Type:Thesis (MRes)
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Abstract

Parkinson’s Disease (PD) is a neurodegenerative disease with limited, symptomatic
treatment options. The gradual progression of PD is characterised by the death of
dopaminergic neurons in the substantia nigra; the slow progression of PD ultimately
resulting in loss of movement and co-ordination is responsible for its high socioeconomic
burden.
A significant barrier to PD research is the lack of animal or cell-based models for the
disease which limits the development of effective treatment. The SH-SY5Y cell line is
frequently used for PD research due to its catecholaminergic phenotype and ease of
maintenance. SH-SY5Y cells can be differentiated to a neuronal phenotype using
differentiating agents including retinoic acid (RA) and Brain Derived Neurotrophic Factor
(BDNF). Cells were differentiated to a neuronal phenotype through the addition of RA and
gradual Foetal Calf Serum (FCS) starvation from the cell media. The process of cell
differentiation was assessed and imaged using inverted microscopy with an attached
camera. The SH-SY5Y cells were successfully differentiated to a neuronal phenotype
using FCS depravation and addition of RA, protocol demonstrated to be consistently
reproducible producing mature, differentiated SH-SY5Y cells within 14 days.
For SH-SY5Y cells to be reflective of the substantia nigra and act as an effective PD
model the cells must be differentiated to a neuronal phenotype; however, many research
papers do not provide information regarding the dopaminergic phenotype of the cells or
explain why the cell line was selected as a model. In this research project we set out to
investigate the reproducibility of SH-SY5Y cell differentiation protocols using RA, to
assess mitochondrial activity of differentiated cells using cell viability assays and to
confirm that differentiated SH-SY5Y cells can successfully synthesise dopamine.
3-(4,5-dimethyldiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and PrestoBlue cellular
viability assays were conducted to assess cellular metabolism and enzyme activity for the
cell population. The ability of differentiated cells to produce dopamine was assessed by
dopamine ELISA.
The results of the MTT and PrestoBlue cell viability assays confirmed that undifferentiated
and differentiated SH-SY5Y cells are metabolically active, however fully differentiated
cells proliferate at a reduced rate, approximately one fifth of that of their undifferentiated
counterparts, indicating that differentiation is stressful to cells and can result in cell death.
ELISA results demonstrate that both undifferentiated and differentiated SH-SY5Y cells
synthesise dopamine with the production being significantly upregulated, by an average of
six-fold, after differentiation.
The findings of this investigation demonstrate that SH-SY5Y cells can be used as an
effective model for PD, providing a suitable differentiation protocol has been used to drive
the cells towards a neuronal, dopaminergic phenotype. However, due to a gap in the
literature the level of dopamine production by differentiated SH-SY5Y cells cannot be
compared against in vivo neurons found in the substantia nigra. Results indicate that SH�SY5Y cells, differentiated by FCS deprivation and addition of RA, can be used as a PD
model however the dopaminergic phenotype of cells should be assessed on a batch-by-batch basis to ensure that cells continue to act as an effective mode

Keywords:Parkinson's disease, neurologic disease, brain, Neurology
Subjects:B Subjects allied to Medicine > B140 Neuroscience
Divisions:College of Science > School of Life Sciences
ID Code:48586
Deposited On:16 Mar 2022 12:41

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