Chin, Ho Wai (Matthew); (2020) Designing platforms to biophysically regulate T cell activation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mechanosensitivity is found in almost every known cell type. While much work has focused on the biophysical regulation of adherent cell behaviours, relatively little is known about the mechanobiology of non-adherent cells such as T cells. The recent rise of cancer immunotherapy has ignited a major incentive for investigating how physical parameters may be harnessed to optimise therapeutic T cell processing. This need stems from the fact that current processing methods are multi-step, labour-intensive, costly and time-consuming. Moreover, T cell activation commonly requires the use of antibody-coated microbeads that lack control over mechanical parameters and require downstream separation. This thesis describes a Nature Inspired Engineering approach to improve T cell processing with hardware platforms for T cell activation. Exploiting T cell mechanosensitivity, a polyacrylamide hydrogel-integrated culture device was developed to stimulate Jurkat T cells with immobilised anti-CD3/CD28. Substrate stiffness and surface ligand density were simultaneously altered to fine-tune T cell activation. Interleukin-2 (IL-2) and post-stimulation proliferation revealed a synergistic effect between substrate stiffness and surface ligand density. Moreover, a trade-off between stimulation strength and post-stimulation proliferation was observed. Relative to stiff hydrogels (62.4  16.7 kPa), soft hydrogels (11.8  3.0 kPa) induced lower IL-2 secretion but higher post-stimulation cell proliferation. Surprisingly, both soft and stiff hydrogels stimulated higher IL-2 secretion than the gold standard T cell-activating material (Dynabeads). Image analysis revealed an underuse of Dynabead surfaces for stimulation due to their heterogeneous dispersion in culture wells. To extend the use of the hydrogel platform, two enabling technologies have been developed for pilot studies – a highly customisable microfluidic system and nanopatterned hydrogels. The former provided a means to automate T cell stimulation under flow conditions, whereas the latter offered gold nanoparticle arrays for spatial control over immobilised ligands. The combination of these tools provide a basis upon which biophysical parameters may be optimised to intensify T cell processing.

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