Développement de systèmes microphysiologiques intestinaux par impression 3D à haute résolution

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The intestine is a complex tissue that is constantly renewed every 3 to 5 days. It is also prone to many diseases. In order to study intestinal homeostasis and the development of these diseases, 3D in vitro models have been developed reproducing several aspects of the intestinal microenvironment. However, current models present some limitations in terms of resolution, manufacturing complexity or materials. Therefore, the main objective of my thesis was to develop intestinal microphysiological systems reproducing the intestinal architecture with materials suitable for the culture of primary intestinal cells and incorporating the mechanical and biochemical aspects of the intestine.The first objective was to generate 3D intestinal structures using stereolithography compatible with cell culture. During this step, I developed a generic optimization protocol for high resolution printing using stereolithography. To do this, I first identified a photosensitive formulation that allows the adhesion and proliferation of an intestinal cell line. Then, combinations of printing parameters were tested and we showed that these parameters influenced the surface properties of the materials, which in turn had an impact on cellular behavior. Simplified intestinal models were then printed with different parameters in order to select the optimal conditions to obtain the desired topography. We characterized the detrimental effect of the absence of oxygen inhibition combined with high light penetration depth, on the printing of cavities. The addition of a photoabsorber to the formulation was necessary to reduce the crosslinking depth and overcome this problem. Finally, the biocompatibility of the printed models was validated by the adhesion, proliferation, and polarization of intestinal cells.The second objective was to adapt the hydrogel previously developed for the culture of primary intestinal cells and, in particular, organoids. As primary cells are more sensitive, the surface of the hydrogel had to be modified to promote cell adhesion. Importantly, a coating was applied and crosslinked to the surface of the hydrogels with an enzyme, transglutaminase, to increase the number of cell adhesion sites. Several key parameters for establishing monolayer organoid cultures on hydrogel have been identified: the composition of the coating and the method of organoid dissociation. In addition, prior enrichment of organoid cultures for stem cells and the use of antioxidants at the seeding improved the culture conditions. Nevertheless, improvements are still in progress regarding the long-term adhesion of the coating to the surface of the hydrogel.The third objective was to develop a microfluidic chip to incorporate the mechanical and biochemical cues of the intestine. A prototype chip was created by 3D printing. This chip consists of two channels separated by a membrane. This chip was designed to enable the direct printing of the intestinal structures onto the membrane using an adapted printer head. This chip will allow the application of shear stress to the cells through the flow of fluids in the channels. In addition, different media will be used in the channels to recreate the intestinal biochemical gradients. Finally, by applying a differential pressure between the channels, the membrane with the structure and the cells can deform and thus reproduce peristaltic movement. Combined with the culture of intestinal organoids, this chip will ultimately allow the generation of intestinal microphysiological systems that will be extremely relevant for numerous studies.

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