Principal Supervisor: Dr. Sophie Cox, School of Chemical Engineering
Co-supervisors: Dr Owen Davies, School of Sport, Exercise and Health Sciences, University of Loughborough; Dr. Federico Alberini, School of Chemical Engineering, University of Birmingham; Professor Liam Grover, School of Chemical Engineering, University of Birmingham
PhD project title: Scalable production of extracellular vesicles
University of Registration: University of Birmingham
Our cells naturally release nano-sized particles, called extracellular vesicles (EV), which participate in a multitude of physiological processes. In the past decade, EVs have emerged as a key cell-free strategy for the treatment of a range of pathologies . Biologically, EVs function is phenotypically dependent, with these particles acting as acellular vehicles for the delivery of a collection of valuable proteins and RNAs with varied and important roles in cell-cell communication, regeneration and disease transmission. As such, if clinically useful numbers of EVs can be generated these nano-particles have the potential to be utilised for a number of significant biotechnological advancements, which includes the development of acellular therapeutics for regenerative medicine or as in vitro biomarkers to predict pandemic diseases such as cancer .
To purify EVs released by cells in-vitro, media is typically collected from tissue culture flasks in batches at defined time intervals. Various methods are then employed to isolate EVs from the culture media, including ultracentrifugation, filtration, precipitation, and chromatography. Currently there is little consensus in the optimal purification approach and poor reproducibility between batches of isolated EVs. Irrespective of the purification method employed, obtaining clinically relevant quantities of EVs is currently a bottleneck in the development of new therapies and diagnostics . To realise the potential benefits of these next generation approaches, there is a need to devise new biotechnology processes to generate large volumes of EVs in combination with efficient as well as consistent isolation protocols.
The overarching aim of this project is to design, manufacture and test novel perfusion bioreactors that allow for concentrated continuous production of EVs at a clinically relevant scale.
This aim will be achieved by completing the following objectives:
Conduct a review of available bioreactors and identify viable systems for EV production
Understanding the hydrodynamic conditions of the identified viable systems and then develop a model to understand the optimal conditions for EV production under varying parameters, which will allow for development of a prototype design
Identify appropriate cell adhesive materials that may be formed at scale into filaments and/or beads which can be incorporated into the bioreactor system
Characterisation of cell adhesion/proliferation using standard assays, test sterilisation of system, and characterise EVs produced
Incorporate identified materials into system design
Conduct a cost analysis of developed biotechnology system to assess economic effectiveness
Develop an appropriate biotechnology manufacturing process for scalable EV production will be achieved iteratively using a combination of modelling, design, manufacture, and biological assays.
This multidisciplinary project will develop key skills in cell culture, industrial biotechnology, modelling, and cost effectiveness. A number of methods will be employed to achieve the objectives listed above:
Literature review and cost analysis of current bioreactor technologies
Computational fluid dynamics modelling
Cell culture and biological assays (viability, proliferation, differentiation)
Polymer processing (extrusion) and characterisation
Characterisation of EVs: size (dynamic light scattering), concentration (NanoSight), composition (transmission electron microscopy), Enzyme-linked immunosorbent assay (ELISA)
Characterisation of hydrodynamic conditions using PIV (Particle Image Velocimetry).
Development of mathematical models to relate yield of EV production to the hydrodynamic conditions
This project will feed into a larger body of work that is focussed on the development of a patent pending (ZSR-1016) EV therapy. It will run concurrently with an EPSRC Landscape fellowship (£250,000 - Davies) and MRC Confidence in Concept funded study (£75,000 – Cox, Davies, Grover) focussed on enhancing the therapeutic effect of EVs for bone regeneration, which involves developing further fundamental understanding of cellular production of EVs and design of an injectable system for an EV therapy. The development of appropriate biotechnology through this project will accelerate the clinical translation of EVs therapy. This strategic approach will elevate the potential impact of the proposed project and it is anticipated that intellectual property will be generated from the optimised bioreactor design and EV isolation system.
- Armstrong, J.P., M.N. Holme, and M.M. Stevens, Re-Engineering Extracellular Vesicles as Smart Nanoscale Therapeutics. ACS nano, 2017.
- Thery, C., Cancer: Diagnosis by extracellular vesicles. Nature, 2015. 523(7559): p. 161-162.
- Watson, D.C., et al., Efficient production and enhanced tumor delivery of engineered extracellular vesicles. Biomaterials, 2016. 105: p. 195-205.
- Quesenberry, P.J., et al., Potential functional applications of extracellular vesicles: a report by the NIH Common Fund Extracellular RNA Communication Consortium. Journal of extracellular vesicles, 2015. 4.
BBSRC Strategic Research Priority: Industrial Biotechnology and Bioenergy
Techniques that will be undertaken during the project:
- Mammalian cell culture and characterisation of viability, proliferation, differentiation (PCR, Immunohistochemistry, flow cytometry, scanning electron microscopy, confocal microscopy)
- Polymer processing: extrusion
- Fluid dynamics: particle image velocimetry, mathematical modelling
- Characterisation of EVs: dynamic light scattering, Nanosight, transmission electron microscopy, x-ray fluorescence, Raman spectroscopy