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Importantly, commonly used in vitro BBB models, such as transwell systems and endothelial cell lines including RBE4, bEnd.3, and hCMEC/D3 monolayers, have important limitations, as they may overpredict permeability and do not fully recapitulate key in vivo BBB features, including physiological shear stress and multicellular neurovascular architecture (Katt and Shusta, 2020)

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