Establishment and Optimization of a Human Flow-Based Hollow Fiber In Vitro Blood–Brain Barrier Model for Systemic Inflammatory Responses

Background/Objectives: Systemic inflammation and circulating proinflammatory cytokines can impair blood–brain barrier (BBB) integrity. Many in vitro BBB models lack the complexity to fully recapitulate systemic inflammation and its long-term effects on the BBB. This study aimed to develop a hollow-fiber flow-based dynamic in vitro (DIV) BBB model for investigating prolonged proinflammatory responses under physiological flow conditions. Methods: Culture conditions for hCMEC/D3 in a DIV model (Flocel) were optimized by varying serum concentrations over seven weeks. Barrier integrity (transendothelial electrical resistance (TEER), permeability studies), proliferation, metabolism (NMR spectroscopy) and molecular changes (high-throughput qPCR) were assessed. Optimized triple-cultures with hCMEC/D3, human primary astrocytes and pericytes were established. After four weeks of barrier establishment, the triple-cultures were exposed to TNF-α, IL-1β and IFN-γ (0.1 ng/mL or 10 ng/mL each) for two weeks. The inflammatory response was assessed with a multiplex cytokine array. Results: Reduced serum concentration (0.25% FBS) decreased proliferation, promoted aerobic respiration, and altered tight junction and transporter gene expression, accompanied by moderately improved barrier integrity compared with 1% or 5% FBS. In optimized triple-cultures, cytokine exposure induced concentration-dependent secretion of IL-6, IL-8, and MCP-1 and changes in mRNA levels, with minor effects on barrier integrity. Sustained cytokine release over two weeks demonstrated stable induction of inflammatory responses at the BBB. Conclusions: An organotypic DIV model of the human BBB, incorporating hCMEC/D3, human primary astrocytes and pericytes was successfully established. By enabling long-term exposure to physiologically relevant cytokine concentrations under flow conditions, this model may provide a platform to investigate functional and molecular BBB responses in inflammation-driven disease progression.