Detachable Modular Blood–Brain Barrier-on-a-Chip with a Concentration Gradient Generator for Stepwise Inflammation Modeling and Drug Evaluation

Background/Objectives: The blood–brain barrier (BBB) is compromised in many neurological disorders, yet in vitro models rarely reproduce graded inflammatory stimulation of brain endothelial cells in vivo. This study developed a detachable, modular BBB-on-a-chip for stepwise inflammation modeling with multi-marker readout, and compared the anti-inflammatory efficacy of quercetin (QUER) and kaempferol (KAE). Methods: The chip combines a Christmas-tree concentration gradient generator with a four-channel culture module, fabricated separately and assembled on demand for stepwise seeding, exposure, dosing, and sampling. hCMEC/D3 cells were exposed to a TNF-α gradient (0–30 ng/mL); extracellular ATP, ROS, NO, cytokines, ZO-1, and ICAM-1 were monitored over 48 h. Transcriptomics and network pharmacology were integrated with molecular docking and 100 ns molecular dynamics simulations; QUER and KAE were administered gradient-wise (0–20 μM), with dexamethasone as a control. Results: The TNF-α gradient induced time- and concentration-dependent injury, with ATP and IL-8 responding at 8 h, ROS and NO at 12 h, and IL-1β, IL-6, and tight-junction impairment later, yielding spatiotemporally resolved readouts across four parallel channels. Ten hub inflammatory genes, including ICAM1, were identified, and network pharmacology nominated QUER. QUER was predicted to bind ICAM-1 more strongly than KAE, its structural analog differing by one B-ring hydroxyl group; gradient administration confirmed QUER suppressed ICAM-1 expression and inflammatory mediator release at lower concentrations. Conclusions: The detachable modular design makes multi-step inflammation modeling and gradient-based anti-inflammatory evaluation convenient on a single chip; QUER’s consistent advantage over KAE across binding calculations and functional assays supports a structure-activity relationship consistent with ICAM-1 engagement.