Design and numerical simulation characterisation of additively manufactured bioreactors for tissue engineering applications

Tissue engineered constructs have great potential in creating representative models of physiology and disease for research and pharmaceutical screening. Dense, mature constructs that mimic the physiology of native disease are required to achieve this. Currently, constructs are limited in size and density by the availability of oxygen and nutrients within the centre of the construct. This limitation could be overcome through the use of perfusion bioreactors which can increase mass transfer into the construct and replicate mechanical cues such as shear stress. The inclusion of tissue engineered models that more accurately represent in vivo physiology in drug screening assays is limited by the bespoke nature of current perfusion systems. Bioreactor models are often produced for specific scaffolds and cannot be easily modified to include multiple cell types and tissue engineered constructs. Additive manufacturing can provide the technology to create complex design geometries and modify bioreactors for the culture of tissue engineered constructs. This thesis aims to develop a perfusion bioreactor system for the culture of tissue engineered constructs that could be rapidly modified for numerous tissue types with a fully characterised fluid flow and mass transfer profile. An existing perfusion bioreactor system printed using selective laser sintering from nylon PA-12 developed by Rimington et al1 was used initially. This bioreactor was used for the perfusion culture of a tissue engineered neuronal construct. This initial work identified limitations in the bioreactor design and operation. The bioreactor produced was porous and unreliable, and the sealing mechanism on the top of the bioreactor was also challenging to achieve. A numerical simulation model was then developed to characterise fluid flow and mass transfer in the bioreactor. No zones of recirculation were present when the bioreactor was operating as designed. This suggested that perfusion culture would support the proliferation of a skeletal muscle construct with no recirculation zones. However, the fluid velocity surrounding the constructs was low and shear stress in the bioreactor was negligible. An increase in fluid velocity did not improve this as the internal geometry of the bioreactor directed flow away from the construct. An area of recirculating media was also found to be present when the top bioreactor seal was not secure. Following this, the physical design of the bioreactor was modified to provide improvements to the bioreactor material, print settings, fixings, and the sealing mechanism. This modified dual construct bioreactor was printed from PLA using fuse deposition modelling. The application of this system was then demonstrated by the continuous perfusion culture of skeletal muscle constructs. This bioreactor was reliable and could be used for the automated culture of constructs. However, no improvements to morphology were seen compared to a static control. The numerical simulation model was subsequently utilised to scale out the bioreactor for the inclusion of six constructs and to develop a homogenous perfusion environment. This was achieved using a vertical perfusion design channelling fluid past tissue engineered constructs. This design achieved a greater fluid velocity adjacent to the construct and a shear stress that could be varied using the inlet fluid flowrate. This research outlines a reliable method of printing a PLA bioreactor perfusion system for the long-term perfusion culture of tissue engineered constructs. This thesis also demonstrates the potential benefits of numerical simulation bioreactor design through optimising a six-construct vertical perfusion design with improved mass transfer and experimental outputs. However, further validation of this system is required. Perfusion bioreactors would benefit from optimisation to both the internal fluid dynamics of the culture chamber and the external design of the bioreactor. Numerical simulations should be utilised to characterise the internal perfusion environment and to optimise fluid flow, shear stress and mass transfer for specific tissue types. Alongside numerical simulations, additive manufacturing has the potential to generate bespoke geometries creating optimised perfusion environments for specific tissue types. Further optimisation of the external bioreactor is required to create simple to use bioreactors suitable for the automated culture of tissue constructs. To create these optimised systems, a multidisciplinary approach is needed with greater access to simulation models.