Scaffolds and signals: design and development of a 3D printed bioreactor and electrospun polymer scaffolds for kidney tissue engineering
Burton, Todd Peter
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There is a pressing need for further advancement in tissue engineering of functional organs with a view to providing a more clinically relevant model for drug development and reduce the dependence on organ donation. Polymer based scaffolds, such as polycaprolactone (PCL), have been highlighted as a potential avenue for tissue engineered kidneys, but there is little investigation down this stream. The focus within kidney tissue engineering has been on two-dimensional cell culture and decellularised tissue. The aim of this project is to utilise electrospun scaffolds within a three-dimensional printed bioreactor system to create an ex vivo environment, to be used as either a conditioning tool for kidney tissue engineering scaffolds or as a model for disease. Electrospun polymer scaffolds can be created with a variety of fibre diameters and the variation in morphology of tissue engineered scaffolds has been shown to affect the way cells behave and integrate. The first study of this thesis examined the cellular response of a kidney cell-line to scaffold architecture using novel electrospun scaffolds. Two fibre diameters were used and three distinct scaffold architectures: random, aligned and cryogenic. The results showed that architecture of the scaffold has a profound effect on kidney cells; whether that is effects of fibre diameter on the cell attachment and viability or the effect of fibre arrangement on the distribution of cells and their alignment with fibres, overall there was a preference for a larger fibre diameter of around 4 μm. Following this, electrospun scaffolds were investigated for their potential to host a multicell population. Rat primary kidney cells were used, and results showed that the scaffolds were capable of sustaining a multi-population of kidney cells, determined by the presence of: aquaporin-1 (proximal tubules), aquaporin-2 (collecting ducts), synaptopodin (glomerular epithelia) and von Willebrand factor (glomerular endothelia cells). Viability of cells appeared to be unaffected by fibre diameter. Overall, the ability of electrospun polymer scaffolds to act as conveyors for kidney cells is a promising strategy for kidney tissue engineering and one that should be explored further; the ‘non-woven path’ provides benefits over decellularised tissue by offering a high degree of morphological control with a scalable fabrication process and a tuneable rate of degradation. Investigation continued with the development of a 3D printed bioreactor. This was a proof of concept device aiming to represent the in vivo environment. The device was designed to be simple to use, delivering a finely controlled shear stress to cells adhered to the scaffold in a dual chamber system, using a modified cell culture media. Computational fluid dynamics was used to gain a better insight into the forces experienced by cells and a modified cell culture media was used to give a better shear distribution across the scaffold, whilst keeping the flow chamber height large. Our investigation demonstrated the ability of the lab scale system to sustain cell life whilst upregulating key transmembrane (AQP-2), cytoskeletal (KRT-8, KRT-18) and tight junction (E-CAD) proteins. Development continued, consolidating the positive characteristics of the bioreactor whilst providing a simplified redesign. In this second-generation bioreactor, the shear profile delivered to each side of the scaffold was mirrored with a flow regime intended to have a low Reynolds number, allowing for the use of two different media without mixing by convection. The device produced a shear stress of 18 to 21 mPa over 80% of the scaffold surface on both superior and inferior sides. The bioreactor maintained a co-culture of endothelial (HUVEC) and epithelial (RC-124) cells, showing the distinct cell types localised on opposite sides of the bioreactor, identified by aquaporin-2 and von Willebrand factor. This bioreactor is a useful tool for modelling of kidney tubules, but has applications in any area where a dual environment with a controlled shear stress is needed. Overall, this work has expanded the breadth of kidney tissue engineering potentially guiding new potential areas of research.