Photopolymerized three-dimensional fiber arrays mimicking the extracellular matrix for the study of immune cell migration

<div> Microfabricated systems offer a powerful means to dissect how local physical cues regulate cell migration and mechanics by providing precise control over geometry, stiffness, and dimensionality. Such control is especially valuable for studying immune cell behavior, as these dynamic cells constantly integrate physical and chemical information. Two-photon polymerization (TPP) has emerged as a versatile tool for fabricating 3D microscaffolds that replicate essential extracellular matrix (ECM) features with submicron precision, high control over geometry, and tunable mechanical properties. Here, we provide a detailed protocol for the use of TPPfabricated 3D fiber scaffolds to study the impact of local microtopography on leukocyte migration and force generation, with a focus on neutrophils, T cells and dendritic cells. The chapter outlines scaffold design, fabrication, cell preparation and seeding, imaging, and quantitative analysis workflows for cell tracking and traction force measurements. This methodology provides a robust framework for probing how specific ECM parameters govern leukocyte motility and force generation, linking migration strategies to physiologically relevant microarchitectures. </div>