Integrating (bio)printing and engineering towards an in-vitro platform to model liver tissue

The liver plays a crucial role in the context of drug development as primarily involved in their metabolism and represents the most frequent reason for drug failure in clinical trials and post-marketing drug withdrawal. Moreover, still efficient treatments are still lacking for liver chronicdegenerative diseases, whose gradual increase in tissue stiffness over the different stages causes chemotherapy resistance. (Bio)printing technique is a promising method in the development of 3D in vitro models to be employed in addressing the afore-mentioned problems. In this context, the following thesis work aims to design a hepatic 3Dprinted in vitro platform capable of covering a window of liver scenarios, ranging from the healthy to different chronic conditions, offering in the same device the possibility of studying new possible drug treatments and understanding more in-depth the mechanisms in a certain hepatic disease of interest. Towards this goal, the conceived system conjugates hydrogels characterized by biological and tuneable mechanical properties with a perfusion device. All these three elements – biological, mechanical, and vascular – are important to be considered in the study of this organ, and, in literature, a 3D model combining all of them is still missing. Each of these aspects has been separately faced. First, we focus on the biochemical part, and we describe two multi-component formulations of hydrogels, to recreate the hepatic microenvironment from a biological point of view. Both formulations contain sodium alginate (Alg), one of the most used biomaterials in bioprinting thanks to its good printing properties, although presenting scarce biological features, and a biomimetic element: decellularized extracellular matrix (dECM) and silk fibroin with lyosecretome. For both biomaterials, alternative preparation protocols, from the ones present in the literature, are proposed and described, followed by their characterization and preliminary biological validation. Then, we address the biomechanical aspect of the model since ECM stiffness has been recognized to have a role in influencing cell behaviour, both in physiological and pathological tissue conditions. The novelty of this part has been the description of an empirical mathematical approach to tailor in advance the stiffness value of the Alg component, whose crosslinking mechanism can be modulated by playing with three factors: Alg concentration, calcium chloride (CaCl2) concentration, and crosslinking time. The whole workflow towards the final obtainment of the predictive equation is shown and validated. Finally, we work on the vascularization element by developing a perfusion bioreactor with the main feature of being versatile in size and shape. We assessed the perfusion system with analytical and computational analyses, fundamental in quantifying two key variables, i.e., shear stress and O2 concentration. The overall study showed the validity of the system, able to sustain a good level of oxygenation, even though with a low level of shear stress at the wall. The system was biologically validated in a preliminary way, showing the improvement of the dynamic condition in the cell culture. In conclusion, the research activities presented in this thesis show, stepby-step, different blocks (i.e., biological, mechanical, and perfusion elements) of a complex puzzle (i.e., the design of a 3D-printed in vitro platform for the liver). To this end, we devote a special effort to combining the concepts of bioprinting with an engineering approach, based on the use of computational and statistical tools, to limit the experimental costly and time-consuming trial-and-error procedures. Each of these pieces has been fully assessed and validated; however, their assembling within the platform concept is still lacking, which will be the goal of future developments.