Conventional migration assays – such as those involving Transwell inserts – fail to recapitulate the structural complexity of tumor ECM. While 3D systems exist, they typically rely on the self-assembly of matrix proteins (e.g., collagen type I), resulting in limited microscale control and reproducibility of the fibrous network. To address this, we characterized tumor-associated ECM networks in BxPC3 xenografts via second harmonic generation microscopy and translated key structural features – such as fiber connectivity and alignment – into 3D printable designs. These were used to fabricate gelatin hydrogel scaffolds at the microscale using two-photon polymerization. However, to study cell migration in a biologically relevant context, these scaffolds need to be integrated into a system capable of generating stable chemotactic gradients.
For this purpose, we have developed microfluidic chips to accommodate the ECM-inspired 3D scaffolds for CAR T cell migration studies. The chip features a three-chamber design, allowing chemoattractant (e.g. CXCL10) to diffuse from a source chamber through a central culture chamber – where CAR T cells are loaded – towards a sink, establishing a chemotactic gradient without the need for external flow. Additional analysis of BxPC3 ECM networks revealed regions with both weak and strong small-world organization. Based on this, representative scaffold geometries were printed directly within microfluidic channels, acting as structural barriers between the culture chamber and chemoattractant source. This platform enables real-time imaging of T cell migration through ECM-like structures under well-defined conditions. By providing a modular, tunable system for studying immune cell navigation in complex microenvironments, this work opens new avenues for evaluating physical constraints on T cell infiltration and guiding the design of next-generation immunotherapies for solid tumors.