Generation of Blood-Brain Barrier-Like Organoids with Improved Endothelial-Cell Encapsulation via Extrusion-based 3D Printing

Abstract

Neurological conditions are the leading cause of disease burden worldwide. Treatment is often complicated by the presence of the blood-brain barrier (BBB), and thus, accurately modeling BBB permeability and penetration is of significant interest. Limitations in 2D in vitro models—particularly the lack of cell interactions—have driven the development of 3D BBB models. However, many 3D techniques face challenges in balancing scalability with complexity. Organoid models offer a simplified platform with greater cell interaction, but they have been ineffective in developing a BBB organoid that is completely encapsulated by endothelial cells–leading to “leaky” BBB models. 3D bio-dot printing has recently demonstrated the ability to generate ECM-encapsulated organoids using a two-step process. Building on this, we propose a technique using two cell-laden inks to print an astrocytic dot within an endothelial dot, forming BBB organoids fully encapsulated by endothelial cells. Our study shows that this dot printing approach can reliably produce BBB organoids with endothelial cells encapsulating astrocytic cores and that these organoids exhibit functional BBB characteristics, including tight junction expression and reduced permeability. However, further investigation of these organoids’ BBB-like functions will be needed to support their potential use in high-throughput analysis of therapeutic-product penetration and substance effect.