Compartmentalized vascular-mimetic glioma spheroid-on-a-chip for modeling endothelial–stromal cell recruitment

models often lack spatial control over tumor spheroids, extracellular matrices, endothelial cells, and stromal support cells. Here, we developed a compartmentalized vascular-mimetic glioma spheroid-on-a-chip for visualizing glioma-vascular-associated cell interactions in a three-dimensional hydrogel microenvironment. The device integrates a central hydrogel channel for pre-formed glioma spheroids, two adjacent medium channels for human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs), and an L-shaped barrier for stable gel-medium interface confinement. Simulation and dye perfusion experiments confirmed improved hydrogel confinement and stable compartmentalization. Optimized HUVEC/NHDF co-culture supported vascular-like endothelial-stromal organization and invasion into the hydrogel compartment. In the integrated chip, the HUVEC/NHDF co-culture increased the diameters of U87 and U251 spheroids by 26.4% and 21.4%, respectively, and induced endothelial-associated cell accumulation around U87 spheroids. Transwell assays further indicated that glioma cells together with stromal cells promoted HUVEC vascular-like organization. This platform provides a simplified and reproducible model for studying selected reciprocal interactions between glioma spheroids and vascular-associated stromal components under spatially controlled three-dimensional culture conditions.