Optimization of a bioink for recreating microbiota crosstalk in mucosal barriers

In recent years, the crucial role of microbiota in human health has become increasingly evident. The human body hosts billions of microbial species, predominantly in mucosal layers such as the gut, lungs, and cervico-vaginal tract. Recent research highlights the interconnection between microbiota from different districts through circulating metabolites. For instance, alterations in gut microbiota have been linked to conditions like cystic fibrosis in the lungs and endometriosis in the reproductive tract, underscoring the need for predictive in vitro models to study these interactions and develop new therapeutic strategies in the fields of probiotics, prebiotics, and antibiotics. Various methods, including animal models, 2D cultures, bioreactors, and microfluidic systems, have been employed to study microbiota. However, these approaches either lack physiological relevance or are too complex for routine use. Hydrogels, 3D polymer structures widely applied in tissue engineering, offer a promising alternative, as they can mimic the mucosal environment where most bacteria reside. This project aims to develop a hydrogel-based bioink for micro-extrusion bioprinting, creating a simple, reliable, and lab-compatible in vitro model that mimics mucosal environments and facilitates inter-district microbiota crosstalk. Firstly, a preliminary feasibility study was conducted to assess the integration of the existing formulation of mucus models for various body districts into a single model. Bioink formulations based on alginate or pectin, tailored for specific districts (gut, lung, cervico-vaginal tract), were developed, and pre-printing evaluations included analysis of rheological properties such as viscosity, storage, and loss moduli, followed by printability assessments based on filament classification during extrusion. Post-printing shape fidelity was evaluated, comparing the bioprinted model to the CAD model. To improve mechanical stability after printing, both ionic crosslinking and photo-crosslinking were evaluated. Photo-crosslinking was aimed, after the inclusion of GelMA and LAP in the bioink. The first strategy showed good results for printable formulations, while the photo-rheology after inclusion of GelMA suggests a possible photo-crosslinking even if not always homogeneous. For this reason, a preliminary development of an alternative photocrosslinking strategy was tested by functionalizing alginate with norbornene, for future thiol-ene reaction and then evaluated through spectroscopy analysis. To assess whether the bioprinted model would be suitable for bacterial survival, oxygen measurement was carried out using UNISENSE technology, showing encouraging anaerobic conditions. To validate the model, preliminary bacterial viability tests using different strains indicated that the printable formulations supported bacterial survival, with some variability depending on the strain and the specific model in which bacteria were printed. Further optimization of alginate functionalization for photocrosslinking, as well as additional tests on bacterial retention time and interactions within the hydrogel, are necessary. However, this thesis, developed within the company Bac3gel, represents a first step in the development of a model to mimic microbiota crosstalk, with future directions potentially including its integration into microfluidic systems or its use as a platform for organoid testing.