Abstract
The loss or failure of an organ is one of the most devastating problems a patient can face.Treatments are extremely limited and commonly involve transplantation with a donor organ,
surgical reconstruction and the use of mechanical devices, such as kidney dialyzers. Since the
1980s, a new field of research at the crossroads of biology and engineering has been developing
novel approaches for those faced with organ failure, termed by Langer and Vacanti ‘Tissue
Engineering’. Their original aims were to develop techniques to produce new tissue, either in
vitro for later implantation, or in vivo by encouraging self-repair. The aims have expanded to
include the development of in vitro models of physiological tissues for disease modelling and
drug discovery. This work will focus on the latter, specifically as it relates to kidney disease and
the glomerular basement membrane (GBM).
The thesis contains a literature review, which covers general approaches to tissue engineering,
3D bioprinting in particular as well as the kidney and kidney disease. This is followed by a
chapter detailing the methods and two results chapters.
In Chapter 3, a reliable tissue engineering system based on 3D bioprinting was developed using
a model cell type, human mesenchymal stem cells (hMSCs). A part-automated methodology for
bioink production was developed and validated using a cell viability assay and electron
microscopy. Next, the previously validated method was applied to conditionally immortalized
glomerular cells. Cell viability was low, and only decreased over time, so a series of experiments
sought to determine the cause. For one of the cell types, glomerular podocytes, two possible
mechanisms were elucidated: anoikis and calcium induced cytotoxicity during crosslinking.
Accordingly, pilot studies using fibrin to promote cell attachment were conducted and showed
improved viability. The final results chapter (Chapter 4) details the application of fibrin to
successful GBM tissue engineering via two distinct methodologies. After validating with hMSCs,
glomerular cells were coated in modified thrombin and bioprinted using the method detailed in
Chapter 3. Though there was evidence of GBM formation, cell viability was still lower than
desired and the cells did not display the morphological changes expected in a bioactive scaffold.
Finally, a novel method leveraging the enzymatic gelation of fibrin by cell membrane–embedded
thrombin resulted in large, stable 3D fibrin scaffolds of glomerular cells and GBM, paving the
way for future use as an in vitro model.
| Date of Award | 26 Nov 2020 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Adam W Perriman (Supervisor) & Gavin I Welsh (Supervisor) |