Exploring the cellular response to acute mechanical force for intracellular delivery and cell therapy applications
File(s)
Author(s)
McCandless, Megan
Type
Thesis
Abstract
As the clinical demand for cell therapies grows exponentially, it is increasingly important that a robust understanding of their manufacturing is provided; from the tools used for intracellular delivery, to the bioprocessing steps employed prior to patient administration. Here, this thesis aims to improve understanding of the cell response to acute, transient mechanical force, which may present as both an inadvertent stressor during bioprocessing steps such as cell sorting, or with the right application, presents as an underexplored opportunity for achieving cargo-agnostic intracellular delivery with minimal cell perturbation.
Across three chapters, two types of mechanical force are investigated: fluid shear stress, and compressive stress. First, a microfluidic platform was used to apply acute, transient shear stress to cells, revealing that across multiple assay readouts, cells are highly robust to shear stress despite significant membrane disruption. This membrane disruption could be tuned by force magnitude and buffer additives, information useful for both intracellular delivery and bioprocessing contexts.
Second, the novel application of a piezoelectric device was investigated as an intracellular delivery tool, using fluid shear stress as the primary mechanism. However, challenges linked to device operating parameters lead to uncontrolled cell lysis. Thirdly, the use of compressive force for intracellular delivery was explored using highly tuneable nanoneedle arrays, where needle geometry and application uniformity emerged as the key determinants of delivery success. The approach offers high spatiotemporal control of delivery, and functionalisation of the nanoneedles with a photo-release mechanism showed promise for further delivery improvements.
Overall, this thesis highlights the challenges and opportunities for using acute, transient mechanical force for intracellular delivery, whilst also establishing useful strategies for mitigating such forces in bioprocessing workflows, ultimately aiming to improve the quality of next-generation cell therapies.
Across three chapters, two types of mechanical force are investigated: fluid shear stress, and compressive stress. First, a microfluidic platform was used to apply acute, transient shear stress to cells, revealing that across multiple assay readouts, cells are highly robust to shear stress despite significant membrane disruption. This membrane disruption could be tuned by force magnitude and buffer additives, information useful for both intracellular delivery and bioprocessing contexts.
Second, the novel application of a piezoelectric device was investigated as an intracellular delivery tool, using fluid shear stress as the primary mechanism. However, challenges linked to device operating parameters lead to uncontrolled cell lysis. Thirdly, the use of compressive force for intracellular delivery was explored using highly tuneable nanoneedle arrays, where needle geometry and application uniformity emerged as the key determinants of delivery success. The approach offers high spatiotemporal control of delivery, and functionalisation of the nanoneedles with a photo-release mechanism showed promise for further delivery improvements.
Overall, this thesis highlights the challenges and opportunities for using acute, transient mechanical force for intracellular delivery, whilst also establishing useful strategies for mitigating such forces in bioprocessing workflows, ultimately aiming to improve the quality of next-generation cell therapies.
Version
Open Access
Date Issued
2023-09-29
Date Awarded
01/06/2024
License URL
Advisor
Stevens, Molly
Sponsor
Medical Research Council (Great Britain)
The Technology Partnership (Firm)
Publisher Department
Materials
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)