Generation of Advanced Blood–Brain Barrier Spheroids Using Human‐Induced Pluripotent Stem Cell‐Derived Brain Capillary Endothelial‐Like Cells

Extensively studied blood-brain barrier (BBB) in-vitro models are established on 2D cell culture inserts. However, they do not accurately represent 3D in-vivo microenvironments due to lack of direct neurovascular unit cellular contacts. Here, the establishment and characterization of a self-assembled 3D BBB spheroid model using human-induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (iBCECs) in combination with primary human astrocytes (ACs) and pericytes (PCs) are reported. This investigation compares 3D spheroids with 2D mono-cultured iBCECs derived from two different hiPSC lines and two differentiation strategies. It is observed that spheroid properties vary depending on the differentiation strategy or type of hiPSC line applied for model generation. However, spheroids demonstrate in-vivo like tight junction ultrastructure and, in comparison to 2D models, higher transcript expression of BBB specific genes. Furthermore, they possess characteristic barrier integrity, barrier functionality, and protein expression. It is inferred that hiPSC-derived BBB spheroids hold a strong potential as a reliable future BBB in-vitro test system.