Engineering a Biomimetic Lab-on-a-Chip Three-Dimensional Ductular Model of the Blood Brain Barrier

Abstract

The blood–brain barrier (BBB) is a highly selective interface that preserves central nervous system (CNS) homeostasis by regulating molecular exchange between the bloodstream and brain tissue. Despite its importance, replicating the structural and functional complexity of the BBB remains a significant challenge in biomedical research. Conventional in vitro and in vivo animal models often fail to capture the dynamic microenvironment and cellular interactions of the native human BBB, highlighting the need for more physiologically relevant and accessible platforms. This study proposes a novel lab-on-chip (LOC) approach to model the BBB using a ductular microchannel design. In contrast to traditional models, which primarily rely on brain microvascular endothelial cells (BMECs), this work employs ECV304 endothelial-like cells as an alternative, due to their ease of culture and ability to form tight junction-like structures. The goal is to develop a simplified yet biomimetic system that improves experimental accessibility while preserving key barrier characteristics. A series of experiments were conducted to evaluate cell attachment, proliferation, and monolayer formation within the microfluidic platform. These included substrate coating comparisons (Collagen I and poly-L-lysine), optimization of seeding density, and the implementation of a chip-flipping technique to achieve dual-sided endothelialization. Cell behavior over time was assessed using fluorescence imaging and quantitative surface coverage analysis. Results showed that Collagen I significantly enhanced cell attachment, yielding up to threefold higher cell numbers compared to control conditions. A seeding density of 20 million cells/mL produced the highest surface coverage of ~40–45% at Day 7, while 10 million cells/mL resulted in a more uniform and stable monolayer formation. The chip-flipping technique enabled successful cell growth on both sides of the microchannel, although some asymmetry remained. Overall, this study establishes a practical framework for developing a microfluidic BBB model using a ductular design and an alternative endothelial cell line, with potential applications in drug testing, disease modeling, and CNS-targeted therapeutics.