Discontinuous dielectrophoresis enabled investigation of high shear stress flows on intracellular calcium signalling of non-adherent cells
Creators: name: Soffe, Rebecca
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310109
subject: Proteomics and intermolecular interactions (excl. medical proteomics)
classificationCode: 310111
subject: Signal transduction
classificationCode: 400899
subject: Electrical engineering not elsewhere classified

description: The opening of Calcium permeable channels in response to their selective stimulus, such as mechanical stress or agonists, causes an influx of Calcium ion into cells. Intracellular calcium signalling is of importance, as the Calcium ion is a potent and versatile intracellular messenger that regulates a wide range of spatial and temporal cellular signals. Intracellular calcium level, is tightly controlled by efflux from the cell through ion transporters, sequestration in intracellular compartments, buffering high affinity cytosolic Calcium-binding proteins, and influx through regulated ion channels. Microfluidic based solutions have assisted in the investigation of various aspects of intracellular calcium signalling, due to the ability for small reagent volumes, parallelism, and short assay durations.<br><br>One area of interest for biologists and engineers is mechanotransduction, in which cells sense mechanical stimulation from their surroundings, which subsequently cause a cascade of biological events in response to this stimulation. These events modulate a range of cellular functions, such as proliferation; differentiation; migration; which in turn are crucial for cellular homeostasis and function. Different mechanical forces, for instance, shear stress, strain/stretch, and compression are being examined on microfluidic platforms, through flow chambers, flexible membranes and valves, respectively. Of interest is shear stress, which if often used for the investigation of drug delivery and transport of chemical agents to target studies, and on the other hand is used as an independent stimulus - to gain a better insight into shear stress flows experienced within the human body. For instance, hemodynamic forces in the Arterial and Venus systems range from 10 to 60 dyn/cm2. However, microfluidic and conventional biological strategies are limited in the shear stress range, which can be applied to non-adherent cells; such strategies are limited to approximately 20 dyn/cm2.<br><br>In order to address this issue, I developed Discontinuous Dielectrophoresis, a novel microfluidic based approach to overcome the limited shear stress range, such that a shear stress range up to 63 dyn/cm2 with a trapping efficiency of greater than 90% can be investigated, as discussed in extensive detail in the thesis. Discontinuous Dielectrophoresis takes advantage of Dielectrophoresis, in which label-free, non-invasive, selective and quick immobilisation of cells can be achieved. However,unlike conventional Dielectrophoresis experiments, the electric field does not remain active during experiments, which allows biological buffers, such as HBS (HEPES buffered saline), to be supplied to the cells during experiments. In addition, the short activation of the electric field, of around 120 s, minimises damage to the cell behaviour and viability – which resulted in cell viability rates greater than 90%.<br><br>In this thesis, I focus on three different experimental shear stress conditions of shear stimulus influence on intracellular calcium signalling, with HEK-293-TRPV4 as the model cell line, transfected stably expressing TRPV4 (Transient Receptor Potential Vaniloid). TRPV4 is a mechano-sensitive, non-selective Ca2+ permeable cation channel, expressed in vascular endothelial cells - which experience shear stress from blood flow. TRPV4 is a polymodal receptor, which plays important roles in cellular processes such as mechano-, thermo-, and osmo- sensation. All different conditions of shear stress simulation were achieved by utilising the Discontinuous Dielectrophoresis procedure, to enable the investigation of high shear stress influence to be investigated.<br><br>Firstly, I investigated the influence of singular shear stress, which occurs in a straight channel, with a singular shear stress present throughout the channel. High levels of shear stress were applied to non-adherent cells, with shear stresses up to 63 dyn/cm2 investigated. With experiments indicating, reduced cellular and peak response times, increased pharmacological efficacy and percentage of activated cells, with increasing shear stress.<br><br>Secondly, I investigated concurrent high shear stress and chemical (TRPV4-selective agonist, GSK1016790A) stimulation. Experiments were undertaken at different shear stress levels and GSK1016790A concentrations. The experiments revealed that shear stress sensitises GSK1016790A-evoked intracellular calcium signalling of cells in a shear-stimulus dependent manner.<br><br>Thirdly, I explored the influence of a spatial shear stress gradient and dynamic shear stress. A spatial shear stress gradient was achieved via the design of a unique lateral trapezoid microfluidic microchannel, which was made possible using 3D printing techniques. As a result the shear stress profile increases laterally across the microfluidic channel, producing a spatially resolved shear stress gradient, over a large shear stress range. Along the spatial shear stress gradient (from low to high shear stress), a decrease in the response and peak response times was observed; however, no change was observed in the maximum fold increase of the intracellular calcium level. Dynamic shear stress, was achieved by varying the shear stress temporally on the trapezoid microfluidic platform; with the temporal shear stress applied in a pulsatile like manner. Experiments revealed that the percentage of cells decreased significantly, and slower cellular and peak response times were observed, in comparison to the spatial shear stress gradient (in the absence of temporal shear stress) of the same shear stress level.<br><br>Consequently, these investigations into different shear stimulus scenarios suggested that the role of TRPV4 may be underestimated in endothelial cells - which experience high levels of shear stress. This in turn, highlighted the importance of conducting studies at high levels of shear stress. In particular, one must also consider more complicated scenarios, such as shear stress gradients, or concurrent stimulation with chemicals or heat, for instance, at high shear stress levels. Or on the other hand, dynamic shear stress, such as temporal shear stress.