Pagliara, Denise (2024) Microenvironment-mediated cell control. A multi-modal microfluidic platform for cardiomyocytes state regulation. [Tesi di dottorato]

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Comprehending the influence of the microenvironment on cell health is crucial for an in-depth understanding of human wellness. This knowledge underscores the necessity of examining how cells adapt and react to their surroundings, particularly through the lens of epigenetic modulation of their behavior. Autophagy, a pivotal epigenetic pathway, plays a significant role in controlling various physiological and pathological processes, including those of the cardiovascular field. The cellular microenvironment in cardiac tissue, characterized by factors such as nutrient availability, mechanical and topographical interaction with the surroundings, is fundamental in the induction and progression of autophagy and to control the consequent cell state. In the context of cardiac health, tissue engineering represents a unique approach to study and potentially modulate cardiac cells wellness. In this PhD work, a microfluidic platform was designed to replicate critical signals of the cardiac microenvironment. A multilayer setup enabled the integration of equi-biaxial mechanical stimulation on a deformable membrane with fluid flow micro-channels for nutrient delivery. Photolithography was used to create topographical cues on the membrane surface, so that a micro-pattern, radially arranged in a circular fashion, was fabricated to align cells in the direction of stretching, mimicking physiological cell arrangement. The device’s functionality was initially verified through COMSOL simulations and subsequently tested with fluorescent labeling to demonstrate equi- biaxial mechanical stimulation and fluid flow interaction. HL-1 rat atrial cardiomyocytes seeded on the platform were able to align with the micro-pattern, migrating along its direction under continuous equi-biaxial deformation. Cells exhibited enhanced beating activity with high coordination upon cyclic deformation on the patterned membrane. Autophagy induction was observed under cyclic deformation coupled with micro-patterned alignment. Notably, enhanced autophagy induction was demonstrated, but, at the final phase of the autophagy flux, a blockage in the fusion and final degradation was observed. However, the patterned arrangement and mechanical deformation significantly enhanced autophagy, as evidenced by a higher total number of vacuoles, compared to the autophagy in non-patterned HL-1 cells. The read-out of autophagy level correlated with a vital cell state, as confirmed by live-dead assays. This suggests that the proposed platform can establish a link between cell state and autophagy under specific cardiac microenvironment signals, such as cyclic mechanical deformation with aligned cell populations. This approach opens avenues for innovative characterizations and therapeutic strategies targeting autophagy pathways, potentially improving treatment outcomes for heart diseases and modifying underlying epigenetic pathways.

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