Promoteur de thèse: Monsieur Sylvain Gabriele
Résumé de la dissertation
While tissue folding observed in vivo in many hollow and tubular tissues can be highly reproducible and robust, folding remains difficult to reconstitute in vitro. In this context, three-dimensional (3D) cell culture aims at reconciliating the simplicity of in vitro models with the human like properties encountered in vivo. Soft permeable hydrogels have emerged as user-friendly materials to grow cells in more physiological conditions. With the intent on turning these homogeneous soft culture substrates into biomimetic templates, we have developed a robust method for manufacturing any desired 3D structures in photopolymerized soft polyacrylamide (PAAm) hydrogels by combining an optical photomask with UV photoillumation. We demonstrated the ease and the versatility of this method by creating simple to complex 3D microstructures in soft hydrogels, as well as corrugations, checkerboard patterns, hemispherical and star-shaped niches. By using atomic force microscopy and laser confocal scanning microscopy, we showed that the elastic modulus of this 3D microstructures can be modulated from 1 to 250 kPa, while keeping the spatial resolution at the micron scale. The cultured of cohesive epithelial tissues on wavy hydrogels showed that matrix corrugations do not affect the overall architecture of epithelial tissues but modulate the orientations of the cell-cell adhesions. Interestingly, our findings indicated that corrugated hydrogels modulate significantly the morphology, distribution and orientation of the nuclei. To further investigate how positive and negative local curvatures of wavy hydrogels lead to important nuclear deformation, we used laser confocal scanning microscopy to quantify the expression of lamin proteins, which are type V intermediate filament proteins enriched at the nuclear envelope. Interestingly, we found that the nuclear A-type and B-type lamins are mechanoresponsive to the substrate curvature. We next studied whether epithelial cells can translate the modulation of matrix curvature into biochemical signals through nuclear deformations. We found that the spatial distribution of Yes associated proteins (YAP), the main transcriptional effectors of the Hippo signaling pathway, is modulated in folded epithelial tissues, whereas negative local curvature zones lead to lower cell proliferation rate and promote significant chromatin condensation. In the last part of this work, we paid attention to one of the many remarkable properties of multicellular tissues, which is their ability to migrate collectively as observed in vivo in wound healing processes or cancer metastasis. We performed time-lapse experiments to explore cellular flows in epithelial tissues grown on corrugated hydrogels. We determined the role of corrugation wavelength on the velocity and persistence of the cellular flows. We found that the tendency for cells to align with the corrugations is the dominant physical parameter that determines the stability of steady flows in the epithelium.