High throughput characterisation of calcium dynamics for single cell functional phenotyping

Kapetanaki, Anna-Maria (2025) High throughput characterisation of calcium dynamics for single cell functional phenotyping. PhD thesis, University of Glasgow.

Abstract

Cell mechanosensitivity is the ability of cells to feel and respond to their surrounding environment, and it plays a crucial role in regulating various physiological processes, including proliferation, differentiation, migration, and apoptosis. Mechanosensitive ion channels are key players in the cellular response to mechanical stimuli. Upon their activation, Ca2+ ions enter the cell and trigger downstream signalling pathways. Therefore, tracking of Ca2+ signalling appears to be a reliable readout of mechanosensitive activity.

However, current methods for mechanosensitivity assessment have low throughput and are not easily applied to large studies or clinical settings. This thesis aimed to address this limitation by creating a device for real-time monitoring of Ca2+ signalling in high-throughput. For this purpose, microfluidics were used as a platform for the device development and cells were stained with a calcium-sensitive dye to facilitate the detection of the calcium influx upon stimulation.

Initially, to understand the operational parameters, the device was tested by chemically stimulating a cell line. Both the chemical stimulant and the cell sample were inserted into the microfluidic device by syringe pumps with a constant flow rate. Upon contact with the chemical stimulant, the cells were probed in different areas of the microfluidic device with a laser source. It was shown that the flow rate and the laser power affect the produced signal, so both experimental parameters should be carefully chosen.

After confirming that Ca2+ signalling could be tracked in real time and with high throughput using microfluidics, newly designed devices were introduced to apply mechanical stimulation to the cells. These microfluidic devices featured constrictions within the channels, allowing cells to be mechanically compressed as they passed through. To assess cellular responses, cells were labelled with a calcium-sensitive dye and exposed to a laser source to enable fluorescence-based detection of Ca2+ signalling. The results showed that both the magnitude and type of mechanical force influenced the cellular response, with those subjected to gentler constrictions exhibiting significantly higher responses than those subjected to more extreme compression.

Finally, stem cell mechanosensitivity and how this is affected by ageing was investigated. Human mesenchymal stem cells were aged on purpose using either physical or chemical methods. Senescent markers confirmed the ageing induction since all senescent cells are aged. Then, the aged stem cells were subjected to mechanical and chemical stimulation to assess their responsiveness. Real-time qPCR and in-cell western tests showed decreased responsiveness to either stimulation. However, some compensatory mechanisms were observed at the posttranscriptional level.

Overall, this study demonstrates that high-throughput microfluidic platforms can effectively monitor cellular mechanosensitivity through Ca2+ signalling. It was shown that both chemical and mechanical stimulations produce measurable responses in cells. The modified responsiveness observed in aged stem cells highlights the critical contribution of mechanosensitive pathways to cellular ageing.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Funding from Engineering and Physical Sciences Research Council (EPSRC).
• Subjects: T Technology > T Technology (General)
• Colleges/Schools: College of Science and Engineering > School of Engineering
• Funder's Name: Engineering and Physical Sciences Research Council (EPSRC)
• Supervisor's Name: Vassalli, Professor Massimo and Dalby, Professor Matthew
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85309
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 07 Jul 2025 15:04
• Last Modified: 07 Jul 2025 15:06
• Thesis DOI: 10.5525/gla.thesis.85309
• URI: https://theses.gla.ac.uk/id/eprint/85309