Development of a Human 3D Immune‐Competent Neurovascular Model Enabling Time‐Resolved Monitoring of Neuroinflammatory Dynamics and Neuroimmune Interactions

ABSTRACT Neuroinflammation disrupts the blood–brain barrier (BBB) and drives neurological disease, but current in vitro MPS and animal models fail to capture its dynamic, human‐specific immune complexity. Here, we introduce a 3D human immune‐competent BBB (3D‐HIC‐BBB) platform that reconstitutes the neurovascular unit (NVU) by integrating primary human brain microvascular endothelial cells, astrocytes, brain vascular pericytes, human microglia, and dopaminergic neurospheroids with embedded transepithelial electrical resistance (TEER) microsensors for continuous functional monitoring. Our platform design enables high‐resolution of inflammatory NVU dynamics that are inaccessible to conventional endpoint assays. Using controlled exposure to tumor necrosis factor‐α (TNF‐α) and lipopolysaccharide (LPS), two distinct triggers of immune responses, we observed stimulus‐specific neuroinflammatory responses with distinct temporal and functional signatures with the resistance full width at half maximum (FWHM) for LPS = 15.8 h and TNF‐α = 4.3 h. TNF‐α exposure elicited a lesser (Δ345 Ω·cm 2 ) but faster drop in resistance and largely faster and reversible recovery than LPS, consistent with transient cytokine‐driven disruption. In contrast, LPS induced a delayed yet more severe (Δ560 Ω·cm 2 ) loss. The 3D‐HIC‐BBB platform distinguishes endotoxin‐ from cytokine‐driven NVU dysfunction and reveals differences in cytokine profiles. It provides a biologically relevant, predictive model for studying neuroinflammation and evaluating anti‐inflammatory therapies.