• 3D bioprinted scaffold
• intestinal in vitro model

In vitro models are currently used for performing studies of physiological, pathophysiological, biological and biomedical nature. Due to their higher repeatability and lower cost, they are considered an alternative to in vivo studies, which pose problems due to ethical implications and lack of translatability. The traditional model used to recreate the mucosa of the small intestine is a two-dimensional monolayer of epithelial cells seeded on the permeable membrane of a Transwell® insert. However, two-dimensional models do not represent tissue-specific architecture, mechanical cues and cell interactions found in vivo. For this reason, numerous 3D in vitro models have been developed in an attempt to bridge the gap between cell culture and living tissue. In that light, this thesis aims at developing an advanced 3D model of the intestinal mucosa using bioprinting techniques.
We designed a villous unit of the intestinal mucosa which was then printed using the INKREDIBLE+ 3D printer. The preliminary tests, in which several printing parameters and bioink composition were optimized, revealed that the chosen approach indeed resulted in the recreation of the villi shape on different supports (glass, polycarbonate Transwell® supports, and polystyrene multiwells).
A bioink composed of 1.1% nanofibrils of cellulose (GrowDex) and 2% alginate was used to print scaffolds, on top of which intestinal epithelial cells (Caco-2 cell line) were cultured for 21 days. During this period, different tests were performed to assess the mechanical and biological properties of the scaffolds. The elastic modulus of the constructs determined through nanoindentation was similar to that of the in vivo tissue (3.23 ± 0.03 kPa after 21 days in culture). The material was shown to be biocompatible, as indicated by the cell metabolic activity assay results and by the formation of the typical epithelial cell monolayer (through TEER measurements and imaging).
Finally, aiming at increasing the complexity of the model, we investigated the feasibility of embedding intestinal fibroblasts in the bioink. The first outcomes indicated that several parameters still needed optimization, but the work had to be interrupted due to the COVID-19 emergency.
Although the cell work needs further development, the context of this thesis was centred on the engineering advancements, rather than the biological studies, and that was successfully accomplished. This work shows that it is possible to achieve a biocompatible structure that better reproduces the intestinal mucosa in vitro through bioprinting techniques, paving the way for the development of physiologically relevant in vitro models.