Abstract
Many potential neurotherapeutic agents fail in the later stages during development due to insufficient blood-brain barrier (BBB) permeability or neurotoxic effects. To address this, we developed an in vitro model incorporating the neurovascular unit (NVU) — astrocytes, pericytes, neurons, and brain microvessel endothelial cells — designed to simulate the in vivo BBB and improve early drug screening. This model uses a direct contact triculture system enhanced by integrating SH-SY5Y neuron-like cells, enabling the study of permeability-linked neuronal responses. Our results show that this expanded NVU model, employing a Transwell® system, enhances the BBB’s restrictive properties and neuronal viability, potentially due to improved cell-cell signaling. Additionally, the model demonstrated increased efflux transporter expression, providing a more physiologically relevant assessment of neuroactivity in relation to BBB permeability. This innovative NVU model offers a predictive and robust tool for evaluating neurotherapeutic agents, facilitating the prioritization of candidates in large compound libraries and potentially reducing attrition rates in drug development. It represents a significant advancement in the methodology for early-stage neurotherapeutic screening, aligning in vitro findings more closely with in vivo responses.