DEVELOPMENT OF A 3D HYDROGEL-BASED MICROFLUIDIC BLOOD-BRAIN BARRIER MODEL WITH ENDOTHELIALIZED MICROVESSELS

Understanding blood–brain barrier (BBB) dysfunction is critical for elucidating mechanisms underlying neurodegenerative diseases such as Alzheimer’s disease. While conventional 2D models are widely used, 3D hydrogel-based systems offer improved physiological relevance, yet dynamic BBB platforms that recapitulate microvascular geometry and integrate multiple cell types remain limited. Here, we developed a hydrogel-based microfluidic device containing a cylindrical endothelialized channel to mimic a brain microvessel. Cell viability was evaluated using SH-SY5Y neuroblastoma cells cultured alone or co-cultured with hiPSC-derived astrocytes (iAstrocytes) within Geltrex™/GelMA hydrogels (1:1 v/v), yielding final GelMA concentrations of 4% or 7.5% (w/v) with Irgacure (0.25% or 0.5% w/v). Softer 4% GelMA promoted enhanced neurite extension and neuronal-like morphology but showed progressive degradation, whereas 7.5% GelMA, particularly with 0.5% Irgacure, provided superior structural stability and sustained viability, especially in SH-SY5Y and iAstrocyte co-cultures, supporting its use for microchannel fabrication. Microchannels were seeded with hCMEC/D3 or hiPSC-derived brain microvascular endothelial-like cells (hiBMECs) at 8 × 10⁷ cells/mL following collagen IV and fibronectin coating. hCMEC/D3 cells adhered within 2 h, formed continuous channel coverage after 72 h, and exhibited low permeability to 4 kDa FITC–dextran, with visually slower extravasation into the surrounding hydrogel than in acellular channels. hiBMECs failed to attach under static conditions but showed adhesion and spreading under dynamic flow, indicating shear-dependent attachment. Overall, this platform supports robust cell viability and endothelialization, representing a promising approach for dynamic BBB modeling.