Puri, J; (2018) Quantitative Investigation into the Relationship Between Substrate Strain and Phenotypic Modulation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

When cells are stretched through substrate strain they respond with changes to their phenotypic behaviour. Given the highly dynamic and mechanically active environment of the human body, this makes sense. In vitro experimentation has demonstrated both this relationship together with the ability to control phenotype using mechanical stimulus alone. Harnessing mechanical stimulus to engineer tissue is thus highly desirable to create new and advance old therapeutics. This thesis questions whether the relationships, as we understand them, between substrate strain and phenotypic modulation are optimal. To build anything new, arguably is it better to know more than you use than to use only what you know. To employ mechanical stimulus as a tool to either create or repair tissue, understanding relationships in general is therefore better. General strain-phenotype relationships, however, are not reported in the literature; dose response type curves do not exists. Arguably this is because current commercial available systems are not designed to do so and to do so would be inhibited by excessive costs. This thesis details the development of a new apparatus conceived to increase the volume of information generated about strain-phenotype relationships with minimal experimentation and costs. The apparatus is based on the principle that the mechanical stimulus delivered to a monolayor of cells, contrary to the majority of apparatus previously developed, is highly heterogeneous. Together with non-destructive assays, image analysis and physical and theoretical modelling, a novel framework to understand these general relationships is sought. To this end, a bespoke 3D printed apparatus is developed which actuates augmented BioFlex plates with small magnets using secondary rotating magnets. The method of actuation is such that it delivers a heterogeneous strain regime across the surface of a BioFlex well. The apparatus is accompanied both by hardware and software to control the periodicity of actuation. Both physical measurement and finite element modelling were utilised to characterise the heterogeneous strain regime across the BioFlex well. Physical measurements were made using digital image correlation techniques offered by Dantec Dynamics GmbH. The technique developed to measure strain deformation on BioFlex plates provides results which concur with intuition and offers a novel alternative to similar strain measurements used on the FlexCell actuation system. Results related to the system developed here highlighted that the variance of strain was not wide enough to suit the ends of the thesis. Finite element modelling was employed to provide a way for the system to achieve the goals of this work as part of future development. The extraction of general strain-phenotype relationships is based upon recording the behaviour of all cells on the BioFlex membrane together with their position on the membrane. A bespoke image acquisition and automated image analysis pipeline is developed which translates cell behaviour across the whole BioFlex membrane into numerical maps for the purpose of statistical correlation studies comparing the equivalent strain stimulus map. Research was also conducted into finding a suitable cell biology paradigm in which the proof of concept apparatus and methodology could be tested within. Experimentation was based upon proliferation studies from the literature however results here are often at odds. Alternative paradigms in the form of plasmid transfection are found to be an avenue for future development. Approaching the need for high-throughput in strain biology experiments through utilising a heterogeneous strain regime as opposed to homogeneous is novel and the development of the bespoke apparatus allows for this idea to be tested further. Characterisation of strain across BioFlex plates utilising digital image correlation has not been done before and appears to be a good high resolution method for physical measurements applicable to other strain inducing modalities, in particular the FlexCell system. The development of a bespoke image analysis methodology allows for tailoring the full pipeline to suit experimental needs and offers an effective way of recording and analysing all cells on a 35 mm BioFlex membrane. Taken together, although the proof of concept is left incomplete, this thesis still makes important contributions to the field.

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