Cerebral Organoids with Integrated Endothelial Networks Emulate the Neurovascular Unit and Mitigate Core Necrosis

Abstract

Cerebral organoids (COs) are multicellular, self-organized, in vitro, 3D brain-like tissues used for developmental biology, disease modelling, and drug screening. However, their lack of vascularity renders them less physiologically accurate. Vascularization of COs remains challenging due to the different requirements between COs and vascular cells, limited vascular network penetration within the organoid, and the absence of luminal perfusion. Here, an encapsulation approach is devised in which human brain microvascular endothelial cells (HBMVECs) are delivered to developing COs from progressively degrading extracellular matrix (ECM)-based hydrogel droplets. By tuning this hydrogel concentration and media composition, an enhanced vascular-like network formation is observed, expanding within the organoid tissue. Using pathway inhibitors, a subset of the endothelial cells (ECs) is shown to originate from the CO itself, promoting network integration. Endothelial networks displayed blood-brain barrier (BBB) features, including astrocytic end-foot-like interactions, pericyte wrapping, and collagen-laminin basal lamina. Vascularized COs exhibited greater media internalization and up to three-fold lower apoptosis than non-vascularized COs. This comprehensive 3D neurovascular model is a promising platform for cerebrovascular research and drug testing applications.

Keywords: angiogenesis; blood‐brain barrier; cerebral organoids; endothelial cells; perfusion; vascularization.