Development of a hydrogel platform to investigate stromal cell mechanoresponse to dynamic stimuli in 3D

Cells and the extracellular matrix exchange mechanical signals, and processes such as breathing and heartbeat periodically deform the matrix. Cell responses to deformation are mostly studied on elastomeric membranes, which reproduce the geometry of epithelial monolayers but not the three-dimensional environment of stromal cells such as fibroblasts. Existing three-dimensional systems rarely combine a quiescent basal state, stable gel anchoring and a single-cell readout. This thesis developed a hydrogel-based platform for static and cyclic stretching of fibroblasts in a three-dimensional matrix. A first two-dimensional configuration confirmed that cyclic stretching activates fibroblasts on collagen-coated membranes, as reported for epithelia. Three matrices were then developed: a physical collagen I/hyaluronic acid gel, a partially covalent variant with methacrylated hyaluronic acid, and a fully covalent gelatin methacryloyl (GelMA) gel. Each required dedicated coupling to withstand swelling and stretching. Polydopamine anchored collagen-based gels via their lysines. In GelMA, by contrast, lysines are consumed by methacrylation, and anchoring required a methacrylate silane and a concentrated photoinitiator primer to counter oxygen inhibition from PDMS. YAP/TAZ localisation showed that collagen I/hyaluronic acid matrices activated fibroblasts regardless of network crosslinking, whereas GelMA preserved a quiescent basal state, a prerequisite for detecting stimulus-induced responses. However, under the conditions tested, the regimen that activated fibroblasts in two dimensions did not activate them in GelMA. This divergence suggests that responses measured on elastomeric membranes may not translate directly to three-dimensional stroma. The platform therefore enables study of fibroblast responses to mechanical stimulation when deformation reaches cells through the surrounding matrix, as in lung, fibrotic and tumour tissue.