Book excerpt: Three-dimensional (3D) cell culture facilitates development of biological relevant assays for drug screening and toxicity testing. Compared to conventional 2D cell culture, cells cultured in 3D can more accurately mimic human tissues and organs and thus provide ex vivo data with potentially better predictive value for cancer research, pharmacology, and toxicology, reducing the need for animal models, improving experimental reproducibility, and reducing time and costs in drug development. The most widely used options for scaffold-based 3D cell culture are, however, based on poorly defined biologically derived extracellular matrix (ECM) with limited possibilities to tailor material properties and that are difficult to combine with state-of-the art biofabrication techniques. The overall aim this thesis was to design and explore modular hyaluronan (HA) based ECM-mimicking hydrogels with tuneable physiochemical properties and biofunctionalities, for development of advanced 3D cell models and biofabrication. The thesis work is presented in five papers. In paper I, we used copper free click chemistry for both hydrogel cross-linking and functionalization with fibronectin derived peptide sequences for culture of human induced pluripotent-derived hepatocytes in a perfused microfluidic system. The tuneable and bioorthogonal cross-linking enabled both retention of high cell viabilities and fabrication of a functional liver-on-chip solution. In paper II, we combined the developed HA-based hydrogel system with homo- and heterodimerizing helix-loop-helix peptides for modulation of both cross-linking density and biofunctionalization. We further demonstrated the possibilities to use these hydrogels as bioinks for 3D bioprinting where both the molecular composition and the physical properties of the printed structures could be dynamically altered, providing new avenues for four-dimensional (4D) bioprinting. In paper III we investigated the possibilities to chemically conjugate full size recombinant human laminin-521 (LN521) in the HA-based hydrogels system using copper-free click chemistry, with the aim to enable 3D culture and 3D bioprinting of neurons. We quantified the impact of using different linkers to tether LN521 and the influence of LN-functionalization on the structural and mechanical properties of the hydrogels. We show that both differentiated and non-differentiated neuroblastoma cells and long-term self-renewing neuroepithelial stem cells (lt-NES) remained viable in the hydrogels. The hydrogels also had a protected effect on lt-NES during syringe ejection and bioprinting. In paper IV, we used HA-based hydrogels modified with peptides sequences derived from fibronectin and laminin for culture of fetal primary astrocytes (FPA). We explored both the interactions between the hydrogels and FPA and possibilities to 3D bioprint FPAs. Finally, in paper V, we developed HA-nanocellulose composite hydrogels with the aim to increase printing fidelity and enable fabrication of multi-layered bioprinted structures without the use of a support bath. In addition to HA, we used wood-fibre derived nanocellulose (NC) to increase the viscosity of the bioink during the printing process. The developed biorthogonal and modular hydrogel systems provide a large degree of flexibility that allows for encapsulation and culture of different cell types and processing using different techniques, which can contribute to further exploration of fabrication of biologically relevant tissue and disease models.