Engineering tools for providing mechanical cues to intestinal in vitro models

Using in vitro cell cultures to study organ-level behavior is a directly scalable and robust approach, but its predictive power is limited by the lack of biological functionality. On the other hand, animal models replicate organ- and multi-organ-level function but are intrinsically defective due to undeniable differences between animal and human physiology. Moreover, they are more expensive than in vitro models and raise ethical issues. There is then the need to develop in vitro models that can provide robust data and, at the same time, replicate organ-level function, obtaining in this way accurately predictive results. This thesis aimed at improving the current intestinal in vitro models, by addressing the physiologic mechanical cues that are missing in traditional static 2D models: a realistic topography of the intestine, a flow of nutrients and oxygen, and the motility of the intestine walls. The PhD project was carried out in the context of a Marie Skłodowska-Curie ITN on electroactive polymers (grant agreement No 641822 "Microactuators - MICACT), thus much of the work was focused on the applications of dielectric actuators for realizing models of intestinal peristalsis. To mimic the architecture of the intestinal villi, a 3D scaffold was fabricated. Poly(lactic-co-glycolic acid) was cast into molds (produced from rapid prototyping methods) and, by employing techniques of thermal induced separation and porogen leaching, porous biocompatible scaffolds were obtained from it. Data from imaging, cell culture and permeability tests confirmed the suitability of these structures to mimic the topography of the intestinal epithelium and to support the culture of intestinal cells. Additionally, the scaffolds were integrated in a dual flow bioreactor and showed that their integrity was maintained during exposure to constant flow for 3 weeks, opening the possibility to include them for an in vitro model where the presence of an adequate flow of nutrients and oxygen is provided to the 3D cell construct. To mimic the motility of the intestinal walls, electroactive polymers were used to construct a stretchable substrate for cells. Progressively refining the characteristics of the produced devices, three bioreactors prototypes were developed. As main outcomes, a planar actuator capable of providing 10% of radial in-plane deformation to Caco-2 cells was established, demonstrating the suitability of the Dielectric Elastomer Actuators (DEA) technology for biomimetic muscle-like actuation. Moreover, after changing the configuration of the DEA actuator, around 5% of equiaxial strain was provided to cultured fibroblasts, inducing changes in the organization of the cell cytoskeleton. The research presented in this thesis illustrates the great potential of exploring both electroactive polymers and microfabrication technologies for the development of biomimetic bioreactors. The outputs of this work can overcome some of the shortcomings of traditional in vitro models and help forging the path towards “ideal” in vitro models which integrate all the cues present in the biological milieu.