HA-based hydrogels for in vitro tumor models

The aim of this thesis work is the development of 3D cancer models through the compartmentalization of hydrogels to mimic different stages of cancer progression. Hydrogels are three-dimensional networks of hydrophilic polymer chains, which absorb high quantity of water. Among different type of biomaterials, hydrogels are widely used as three-dimensional supports to cell culture thanks to their structure similarities to extracellular matrix (ECM) of mammalian tissues. In this project, we designed 3D in-vitro tumour models by recreating both the stroma and tumour compartments. For the stroma compartment, we developed hyaluronic acid (HA)-based hydrogels with tuneable stiffness, which were embedded with fibroblasts. Then, we integrated tumour compartment though 3D bioprinting of breast cancer cells within the hydrogels, encapsulated in a bioink. This distinct compartmentalization enables us to investigate how matrix stiffness influences the migration, invasiveness, and morphology of cancer cells, as well as how fibroblasts rearrange in response to these changes, thereby modelling different phases of cancer progression. To formulate a tuneable stromal matrix, we firstly study the effect of incorporation of chitosan into the HA-based formulations. We focused on developing HA-chitosan systems with tuneable stiffness by varying the molecular weights and concentrations of the polymers. However, these systems resulted in a viscoelastic liquid rather than a gel or formed dense precipitates due to the strong electrostatic interactions between the oppositely charged biopolymers. Consequently, this process limited our ability to tune stiffness. To address this, we incorporated type I collagen into the HA-chitosan formulation while reducing the concentration of chitosan. Given the reduced amount of chitosan, we ultimately developed a formulation combining hyaluronic acid (HA) and collagen, enabling stiffness adjustment by varying the molecular weight of the HA, which was assessed through rheological analysis. For the recreation of the tumour niche, we used extrusion bioprinting technique to print bioinks made of Matrigel containing high-density suspension of breast cancer cells. Adjusting key parameters of extrusion (nozzle diameter, pressure, speed, cell concentration), a single drop of bioink is printed inside the HA-based hydrogels, thus realizing the spatially controlled compartmentalization of hydrogels. In this way we could mimic the heterogeneity of cancer microenvironment, enhancing the study of cancer growth and progression. Although in-vitro cancer models are simplifications of the in-vivo scenario, they provide valuable insights into the key characteristics of the natural microenvironment, facilitating the investigation of cell-cell and cell-extracellular matrix (ECM) interactions. Ultimately, these models serve as tuneable platforms to investigate the relationship between the mechanical properties of the tumour microenvironment and the spatiotemporal evolution of cancer growth.