Saporito, Stefania (2023) Time and space modulation of substrate curvature to regulate cell mechanical identity. [Tesi di dottorato]

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Abstract

Each cell can sense and transform external mechanical stimuli into internal chemical reactions thanks to two cell’s abilities known as mechanosensing and mechanotransduction, respectively [1]. The mechanical cues can be either extracellular forces applied on the cell from the extracellular matrix (ECM) such as compression, tension and fluid shear stress, or intracellular forces like those arising from cellular responses to changes in extracellular matrix stiffness. These mechanical stresses could control cell shape, cell proliferation, cell polarity and cell differentiation; furthermore, in pathological conditions, such as cancers or neurodegenerative diseases, the mechanosensing and mechanotransduction pathways result different from normal cells [2, 3]. On the cell scale, the geometric form and the biological functions are inherently correlated and there is also increasing evidence of the effects of substrate curvature on cell behaviour, even if these effects are largely overlooked or underestimated during the design of new cell-material interfaces [4]. Moreover, there is a spatio-temporal dependence in the influence of cell response from its surrounding environment which has to be considered [4]. In fact, in the body, the cells especially those from blood, bone marrow, lungs, heart and musculoskeletal tissues are subjected to cyclically mechanical loading. To mimic such dynamic mechanical microenvironment, the controlled stress/strain could be applied to cell-seeded biomaterials designing specific microfluidic devices. Different microfluidic platforms have been designed to analyse the cell responses to static [5-8] or dynamic mechanical loadings [9-13], but in all these studies, the effects of these solicitations are viewed at the end of stimulation and/or on confluent cells. Conversely, a real-time analysis on single live cells is necessary to disclose the role played by these mechanical cues on cell fate and behaviour. So, the aim of this project is to design a microfluidic platform where single adipose derived mesenchymal stem cells (ASCs) are statically or dynamically stretched modifying substrate curvature and then, their response is acquired in real time using the confocal microscopy technique. The real-time visualization with a confocal microscope is possible using the transfection technique, which here has been obtained by electroporation strategy. Detailly, to study the response of single adipose stem cells to a static/dynamic curvature solicitation, the structural changes of the actin cytoskeleton and the focal adhesions (FAs) have been explored and quantified. Moreover, the actin cytoskeleton can convert this mechanical stimulus into biochemical responses. Specifically, the actin cytoskeleton can directly regulate the activation of Yes-associated protein (YAP), a transcription factor implicated in cell proliferation, differentiation, and survival [14]. It is not well understood the role of curvature in the regulation of this specific transcription factor, particularly for concave surfaces. In fact, the convex substrates should be able to activate the YAP translocation from the cytoplasm to the nucleus and then, induces cellular proliferation [15,16]. Conversely, a correlation between concave morphologies and tight junctions for high-density cells should be responsible for YAP shuttling from the nucleus to the cytoplasm but this mechanism has not completely elucidated [17-19]. So, the designed microfluidic platform has been used to study the mechanotransduction response to a specific curvature variation in terms of YAP translocation (from the nucleus to the cytoplasm and vice versa) in ASCs cultured at high-density. The designed microfluidic platform is able to induce macroscopic morphological topographies to adipose derived mesenchymal stem cells which are able to finely control actin stress fibers, focal adhesions, and YAP responses. Conversely, micro and nanoscopic mechanical features could be applied by designing specific patterns of hundreds of nanometres on the PDMS cell-seeded membrane of the chip. Being micropatterns characterized by morphological featured within the same order of magnitude of cell receptors, they are able to act at level of focal adhesions. So, a specific micropatterned substrate has been designed and fabricated using the two-photon polymerization technique which will be used as master for the PDMS membrane manufacture. In this way, the microfluidic platform can be considered as a tool to apply different mechanical insults on ASCs in a simultaneous manner. This work is organized as followed: the theoretical background regarding the cell structures involved in mechanosensing and mechanotransduction pathways, the role of microenvironment curvature on the cell behaviour and state of the art about the design of microfluidic platforms as tools for cellular mechanical stimulation are given in Chapter 1, establishing relevant definitions for a sufficient background knowledge for the work presented in this thesis. The Chapter 2 describes the design of a new microfluidic platform to stimulate in a static or dynamic way adipose derived mesenchymal stem cells and analyse their response in real time combining microfluidic device – confocal microscopy technique – electroporation strategy. In the Chapter 3 the actin cytoskeleton organization and focal adhesions behaviour in static mechanical configurations have been analyzed; conversely the dynamic mechanical response of the same structures has been observed in Chapter 4. Then, the response of YAP to a specific curvature morphology and the design and fabrication of a specific micropatterned substrate were amply explained in the Chapters 5 and 6, respectively.

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