Development of the novel BRB model utilizing hiPSC-derived cells

The inner blood-retinal barrier (iBRB) is a complex structure crucial for ocular health, involving interactions between endothelial cells (ECs), pericytes (PCs), and Müller glia cells (MGCs). Because the iBRB is essential for preserving retinal homeostasis and preventing pathogens from penetrating the retinal tissue, its impairment is linked to several ocular diseases such as diabetic retinopathy, age-related macular degeneration, retinal vein occlusion, uveitis, etc. Accurate in vitro modeling of the iBRB is essential for understanding retinal diseases and developing effective treatments. This study aimed to establish and analyze an in vitro model of the iBRB using monoculture, direct, and indirect co-culture methods to investigate the roles of ECs, PCs, and MGCs in barrier formation and function. The project was divided into two main approaches: indirect and direct co-culture of ECs with PCs and MGCs. In indirect co-culture, ECs were first cultured on 24-transwell (TW) inserts, followed by co-cultivation with NCPCs and MGCs in 24-well plates. For direct co-culture, ECs were seeded on 24-TW inserts, and PCs and MGCs were seeded on the opposite side of the membrane. Immunofluorescence microscopy, immunocytochemistry, and cryosectioning were employed to assess cell morphology, protein expression, and interaction. Permeability was tested using FITC-dextran of varying molecular weights. In indirect co-culture, ECs exhibited full confluence with minor influence from MGCs. The permeability tests revealed no significant difference in barrier integrity between control ECs and those co-cultured with NCPCs or MGCs. In direct co-culture, PCs might enhance the maturity and organization of the EC monolayer, as indicated by increased cell density, elongation, and intercellular junction formation. MGCs had a less pronounced effect on EC morphology. Immunocytochemistry showed complex ECM patterns and distinct protein expressions, such as CD44 and PDGFR, in different co-culture conditions. This implies that pericytes might stimulate Col IV expression in ECs, whereas MGC might have a lower or inhibitory effect on Col IV expression in ECs. Our study demonstrates that the established direct and indirect co-culture methods can be used to model various aspects of the iBRB. We show the different effects of PCs and MGCs on the EC monolayer and barrier function, confirming the different roles of each cell type in maintaining the iBRB. This model provides a valuable tool for understanding the cellular interactions within the iBRB and their implications in retinal diseases and drug development.