Cellular mechanotransduction refers to the process of converting external mechanical stimuli into internal biochemical signals. This conversion mediates many cellular functions where defects in mechanotransduction can lead to the initiation and progression of disease. Given its importance, a technology capable for activating mechanosignalling via application of mechanical stimuli to investigate and screen mechanotransduction in either two-dimensional (2D) or three-dimensional (3D) context can provide a powerful tool for basic cellular studies as well as identifying potential/repurposing current therapeutic compounds.
Here, we present the development of a high-throughput optical technology known as the μTsunami platform that utilizes a pulsed laser microbeam to mechanically perturb cells by microcavitation bubble (μCB) generated impulsive fluid shear stresses and standard fluorescence microscopy for evaluating the resultant cellular mechanosignalling. We establish the capability of μTsunami induced shear stress impulses to activate cellular mechanotransduction. This was confirmed via μTsunami exposure of primary adherent human umbilical vein endothelial cells (HUVECs) plated on fibronectin coated glass-bottomed dishes which led to initiation of Ca2+ signalling. Moreover, we demonstrate the capacity of our platform to accurately measure suppression of Ca2+ mechanosignalling in a dose-dependent fashion when putative inhibitors were administered and completed a mock high-throughput screening (HTS) experiment.
We hypothesize μTsunamis initiate Ca2+ mechanosignalling by mechanical stimulation of stretch-sensitive G Protein-Coupled Receptors (ssGPCRs) which activate the IP3 pathway. Ca2+ signalling due to diffusible factors released by cells proximal to the μTsunami which turn on purinergic receptors remains an alternative postulate. To differentiate between these two hypotheses, we conducted studies to investigate the effect of chemical inhibitors of key molecular proteins along the IP3 pathway and purinoceptors on μTsunami-initiated mechanosignalling. Our results demonstrate that μTsunami-induced Ca2+ signalling in HUVECs was activated mechanically and does not arise via the chemical activation of purinergic receptors. Moreover, we determined the spatial extent of Ca2+ signalling is dependent on the magnitude of shear stress impulse that the cells are exposed to independent of laser microbeam pulse energy. This establishes a clear mechanical dose-response relationship for μTsunami activated mechanosignalling.
Lastly, we introduce a non-invasive technique to measure the viscoelastic properties of soft matter at high strain-rates known as Laser-Induced Cavitation Rheology (LICR). LICR utilizes experimental measurement of cavitation dynamics within hydrogels, theoretical prediction of bubble dynamics using a viscoelastic model that accounts for potential material failure, and retrieval of material properties using non-linear least squares optimization. For biologically and synthetically derived hydrogels, we demonstrated LICR not only is capable of quantifying the maximum cavitation radius ???? and elastic moduli ? as well as the strain at which the viscoelastic material fails ??. Furthermore, we presented preliminary evidence of mechanically activating Ca2+ signalling in HUVECs embedded within fibrin gels which demonstrates the ability of our technique to apply physical stimuli in a 3D context.
Collectively, these results effectively establishes our technologies in providing mechanical perturbation in both 2D and 3D context for the investigation and screening of cellular mechanotransduction. This platform presents unique opportunities for the investigation of mechanosignalling pathways and characterization of materials at high strain-rates.