• ADSCs
• agarose hydrogel
• dextran
• epsilon-dot method
• lumped parameters
• mechanical properties decoupling
• mechanotransduction
• stem cells
• visco-transduction

The aim of this work of thesis was to identify a method for engineering viscoelastic properties of hydrogels in their use as culture substrates in the study of visco-transduction, i.e. the study of cellular response to viscous stimulations. As a result of the key role played by mechanotransduction in directing and controlling cellular behaviour, several studies have focused on cellular response to stiffness. Despite the intrinsic viscoelastic nature of biological tissues, it was not until recently that cellular response to viscoelasticity has started to be investigated. These studies highlighted the importance of designing in-vitro models capable of preserving stem cells multipotency. The relationship between cellular behaviour and the viscous component of the culture substrate has generated great confusion among scholars, which prompted the need to design a simple and reproducible method which would make it possible to engineer the viscoelastic properties of hydrogels, by focusing mainly on the modulation of their viscosity. This work of thesis focused on the design of a method which could modulate the viscous properties of agarose substrates by adding dextran to the aqueous phase, while maintaining a constant equilibrium elastic modulus. Viscoelastic parameters were evaluated by means of the Epsilon-dot method. Mechanical characterization was supported by computational models for the analysis of the behaviour of hydrogels during their liquid phase in the presence of dextran, as well as the interaction between the polymer network and the engineered liquid phase. The first model implemented a reaction-diffusion equation which described the liquid phase flow during the compression test, while a finite element method (FEM) model was implemented to analyse the dextran flow outside the agarose hydrogel during the cell culture.
It was demonstrated that not only does dextran behave as modulator of the liquid phase viscosity, but it also functions as a weakening agent of the bonds between water and the agarose polymer network due to the decrease in instantaneous elastic modulus and relaxation time. As shown by the mathematical models, this results in the greater flow of free water with a relative increase in the diffusion coefficient. In order to adapt the engineered hydrogels for cell culture, 0.5% w/v agarose hydrogels were identified to better mimic the adipose-derived mesenchymal stem cells (ADSCs) niche, which has a stiffness between 2 kPa and 6 kPa. The optimal culture conditions were established and 5% gelatin coating and 50.000 cell/cm^2 were identified as best working conditions. Finally, it was determined that ADSCs culture for the study of visco-transduction in preserving cell stemness should not last longer than 21 days. The immunostaining of YAP and CD45 highlighted that YAP was localised inside the nucleus after 21 days, while CD45 was higher than 2%, thus cells seeded on TCP were losing their stemness after 21 days.
The final goal of this work was to define an optimal set-up for the study of ADSCs response to visco-transduction, in order to define more physiologically-relevant culture conditions and to control cellular response for the design of in-vitro models and for regenerative medicine applications.