Speaker
Description
Introduction
Depression affects over 350 million individuals globally, with 20–30% developing treatment-resistant depression (TRD), a major contributor to suicide risk. Existing preclinical models inadequately recapitulate the complexity of the human neurovascular unit (NVU) and blood–brain barrier (BBB), thereby limiting the advancement of effective therapeutics. The objective of this study was to develop a three-dimensional (3D) BBB model to enable mechanistic investigations of barrier permeability, neuroinflammation, and pharmacological responses.
Methods
A two-channel polydimethylsiloxane (PDMS) microsystem separated by a porous polycarbonate membrane was fabricated. The lower channel was employed to reconstruct the BBB using the Double-Viscous Finger Patterning (Double-VFP) technique: human brain vascular pericytes (HBVP) and astrocytes (HBVA) were embedded at a 1:3 ratio within a 5 mg/mL collagen I hydrogel to form the lumen, followed by seeding of human brain microvascular endothelial cells (HBMEC) at a 3:1:3 ratio. Culture conditions were optimized for a 10-day period. Cellular viability (AlamarBlue® assay), dextran permeability, CellTracker®-based imaging, and immunostaining analyses were conducted on days 1, 3, 7, and 10; 3D imaging was performed at 24 and 48 hours post-seeding. Cellular morphology within the hydrogel matrix was compared to traditional two-dimensional monolayer cultures via immunocytochemistry to validate model fidelity. To simulate an inflammatory environment, hormonal stimulation using cortisol alone and a combination of cortisol, aldosterone, and angiotensin II—molecules known to be elevated in depression—was optimized in macroscale models employing AlamarBlue® and RealTime-Glo™ MT Cell Viability assays. The upper microchanel allows for the integration of neurons to create together the NVU model (under development).
Results
The microsystem enabled reproducible generation of single- and double-lumen structures, with future compatibility for the incorporation of neurons and microglia. The Double-VFP method supported sustained high cell viability over 10 days, with the formation of a continuous and functional endothelial layer. Relative to the Single-VFP approach, the Double-VFP technique facilitated more rapid organization of cellular components into a functional BBB and exhibited superior barrier integrity, as evidenced by dextran permeability assays. Complete assembly of the BBB architecture was confirmed by CellTracker® imaging and immunostaining within 48 hours. Hormonal stimulation at both low and high concentrations altered cellular metabolic activity, indicating a biological effect that will be corroborated with additional assays, such as ELISA.
Discussion
The developed 3D BBB model—particularly utilizing the Double-VFP method—successfully replicates key physiological features of the BBB and provides a dynamic platform for the study of barrier permeability, neuroinflammatory processes, and pharmacological testing. Its rapid and reproducible assembly, coupled with its structural and functional robustness, positions it as a valuable tool for mechanistic studies in neuropharmacology and for the preclinical evaluation of psychotropic, psychedelic, and anti-inflammatory compounds. Ongoing integration of iPSC-derived neurons and microglia is anticipated to further enhance the model’s relevance for the investigation of depression pathophysiology and therapeutic development.
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