"Introduction: The burden of central nervous system (CNS) diseases continues to increase especially with an aging global population. The interface between the brain and the blood is governed by the specialized endothelium which composes the blood-brain barrier (BBB). Brain metastasis has a high rate of incidence from melanoma, breast and lung cancers and the BBB is strongly involved in these events. The breakdown or malfunction of the BBB plays a pivotal role in CNS diseases. Moreover, drug delivery to the brain is often hampered by the tight BBB. This study aims to develop a biomimetic BBB model that closely mimics the BBB physiology to investigate drug delivery and cancer metastasis to the brain.
Methods: The BBB model was created by co-culturing human aortic endothelial cells on top of human astrocytes encapsulated in gelatin-methacrylate (GelMA) hydrogels. The astrocytes were encapsulated in GelMA hydrogels of different concentrations (5%, 7.5% and 10% w/V) to study their growth and proliferation in a 3D environment. The viability of astrocytes was assessed via live/dead assay. The morphology of the astrocytes was evaluated by fluorescent imaging. The endothelial cells were cultured on top of the hydrogels on inserts having 8 µm pores where endothelial cells cultured on inserts with 0.4 µm pores with astrocytes cultured on the bottom side were used as controls. The barrier function was studied by transendothelial electrical resistance (TEER) and Evan’s blue albumin (EBA) permeability assay. Tight junction protein, ZO-1 formation of the endothelial cells was assessed by immunofluorescent staining.
Results: Astrocytes encapsulated in 5%, 7.5% and 10% GelMA hydrogels exhibited a high viability (>80%) with no statistical significance between the different concentrations. Encapsulated astrocytes had a star-shaped morphology in the hydrogels. TEER measurements of endothelial cells cultured on GelMA hydrogels encapsulated with or without astrocytes showed higher values when compared to the plastic counterparts. Despite higher TEER values, the permeability of EBA were higher in the hydrogel models. This is due to the different pore size of the inserts used in the hydrogel models which only allowed ~3% of EBA to cross when compared to an empty insert while the plastic counterparts allowed ~ 15%. The ZO-1 staining showed more tight junction formation in the GelMA hydrogel models with or without astrocytes.
Conclusion: This study utilizes biocompatible GelMA hydrogels which provide a 3D environment for the growth of astrocytes. The hydrogel model provides insight into the effect of extracellular matrix on cell growth and function. The hydrogel model will allow the studies of cancer invasion and migration required to investigate the interaction of the BBB with metastatic cancer cells. The 3D biomimetic BBB model provides a tool to investigate brain metastasis and the administration of drugs to the brain with high fidelity."