Human organ chip models recapitulate FRC-driven high endothelial venule differentiation, immune recruitment, and cancer-induced disruption.

High endothelial venules (HEVs) are specialized post-capillary venules that gate lymphocyte entry into lymph nodes, yet human in vitro models that capture their specialization remain limited. Here, we develop a perfusable human HEV-on-chip in which blood endothelial cells (BECs) form an engineered microvessel within a collagen-fibrin matrix populated by human fibroblastic reticular cells (FRCs). The presence of FRCs, without exogenous LIGHT, drives HEV-associated marker expression (PNAd/MECA-79 and VCAM-1) and a plump endothelial wall enwrapped by a thickened laminin+ basement membrane, recapitulating features of lymph-node HEVs. Functionally, HEV-like vessels support markedly increased T-lymphocyte adhesion while preserving VE-cadherin junctions. Introducing MDA-MB-231 breast cancer cells in an adjacent channel reduces MECA-79, disrupts junctional organization, and increases permeability without inducing apoptosis. Together, this platform defines a stromal instruction program for human HEV identity and a tractable model of cancer-associated HEV dysfunction.