A Biomimetic Human Multi-Cellular In Vitro Model of the Blood–Brain Barrier

Current in vitro models fail to recapitulate specific physiological properties of the human blood-brain barrier (BBB); hence the need for a reliable platform to study central nervous system diseases and drug permeability. To mimic the normally tight blood-brain interface, primary human endothelial cells (HAECs) and primary human astrocytes (A) were grown in a confined space of the physical scaffold created by gelatin methacrylate (GelMA) hydrogel to allow optimal astrocyte-endothelial cell direct/indirect interaction. Evidence for a physiologically relevant BBB was established by assessing the expression of tight junction markers conferring the barrier function, and by measuring biophysical attributes using the trans-endothelial electrical resistance (TEER) and the Evans blue albumin (EBA) permeability assay. An HAEC+A three-dimensional (3D) co-culture was associated with 12-fold higher <i>claudin-5 (CLDN5)</i> and <i>cadherin-1</i> (<i>CDH1</i> or <i>Epithelial [E]-cadherin)</i> transcriptional levels than two-dimensional (2D) models. This model conferred the highest TEER (45 Ω·cm²) in 3D HAEC+A, which value was 30 Ω·cm² in 2D (<i>p</i> < 0.01) and 25 Ω·cm² in 3D HAEC cultures (<i>p</i> < 0.001). Functionally, in 3D HAEC+A co-cultures, higher TEER resulted in 10-fold and 7-fold lower EBA permeability at 120 min, in HAECs alone or in to 2D co-cultures (<i>p</i> < 0.01). The established human primary cell model has acquired features mimicking the human BBB in vitro, and is now poised to be tested for the permeability of the BBB to pharmacological agents, parasites, cells (such as brain-tropic cancer cell metastasis) and any mechanisms that might involve traversing the BBB.