Nanocellulose-based hydrogels for 3D cell culture

In the last few years, research is focusing on the synthesis of materials that are mimetic of the natural extracellular matrix (ECM) and thus able to provide cells with the biochemical and mechanical signals necessary for their growth and proliferation. Cells of all tissues are surrounded by and interact with this three-dimensional soft extracellular matrix (ECM) and from it they receive physical and biochemical stimuli that play a major role in regulating cell behaviors. Nowadays, the gold standard for 3D culture is Matrigel, however it has several undeniable drawbacks. Matrigel is extracted from the Engelbreth–Holm–Swarm mouse sarcoma, and since is animal-derived, it is affected by a large batch-to-batch variability in addition to a potential risk of transmission of animal pathogens. The aim of this thesis work is to design a synthetic matrix suitable for 3D cell cultures that is totally defined and tunable and that could replace and overcome the main limitations of Matrigel. Among the different materials that can be used for this purpose, cellulose was chosen because nanocellulose hydrogels are highly hydrated soft materials with good mechanical properties. These cellulose-based gels can be produced from bacterial or plant cellulose nanofibrils (CNF), which are renewable, biodegradable, and biocompatible. As a raw material, it possesses the inherent limitation of being hydrophobic; however, owing to the numerous OH groups present in its structure, it can be easily functionalized with hydrophilic groups to overcome this limitation. In addition, cellulose fibers have a high surface area and can be chemically modified with functional groups or by grafting biomolecules. Cellulose functionalization provides enhanced physical and chemical properties and control of biological interactions, tailoring its hydrogels for specific applications. The synthesized material was then characterized chemically and structurally, by means of conductimetric titration and dynamic light scattering (DLS) analysis. Moreover, the gel mechanical properties were studied, by means of rheological analysis, and finally biologically, by means of preliminary cell embedding to follow the cells’ growth and expansion. Therefore, and because of its defined chemical composition and tunable properties, the CNF hydrogel presents a viable alternative to the growth and development of 3D cell cultures such as spheroids or organoids with the option for clinical use, however, further optimizations are required to achieve the maturation and growth achieved in Matrigel.