Résumé
SignificanceCells evolve within intricate, fiber-rich microenvironments that present significant challenges for studying their behavior and mechanics. To address this, we employed advanced three-dimensional (3D) microfabrication to create parallelized arrays of fibers with controlled geometry and mechanics that can be deformed by cellular forces. This system is integrated with an automated pipeline to measure these forces in three dimensions. We validated the platform using various models, from connective tissue or blood vessel cells to immune cells, demonstrating its ability to analyze a wide range of cellular dynamics and adhesion forces. By replicating fibrillar environments with tunable architectures and mechanics, this approach provides a powerful tool for characterizing cell mechanics, offering valuable insights into cell interaction with complex, customizable mechanical landscapes.
The forces exerted by cells upon the fibers of the extracellular matrix play a decisive role in cell motility in physiopathology. How the local physical properties of the matrix (density, stiffness, orientation) affect cellular forces remains, however, poorly understood. Existing approaches to measure cell three-dimensional (3D) traction forces within fibrous substrates lack control over the local properties and rely on continuum approaches, not suited for measuring forces at the scale of individual fibers. Herein, an approach is proposed to fabricate multilayer arrays of suspended deformable fibers spanning a wide range of fine-tunable geometrical and mechanical properties using two-photon polymerization. Atomic Force Microscopy is used to thoroughly investigate the properties of individual fibers, including Young’s modulus and stiffness. This approach is combined with a reference-free method for measuring traction forces in 3D, which relies on automated segmentation of the fibers coupled with finite element modeling. The force measurement pipeline is applied to study forces exerted by endothelial cells, fibroblasts, or macrophages, and reveals how these forces are influenced by fiber density and stiffness. Additionally, coupling to fast volumetric imaging with lattice light-sheet microscopy enables the measurement of the low-intensity and short-lived tractions exerted by amoeboid cells, such as dendritic cells. Our technology will be instrumental for monitoring and studying cell behavior at the single-fiber level at extracellular matrix density interfaces, which play a crucial role in both physiological and pathological contexts, such as tumor boundaries.