title: Organization of Endothelial Cells, Pericytes, and Astrocytes into a 3D Microfluidic in Vitro Model of the Blood-Brain Barrier.
abstract: The endothelial cells lining the capillaries supplying the brain with oxygen and nutrients form a formidable barrier known as the blood-brain barrier (BBB), which exhibits selective permeability to small drug molecules and virtually impermeable to macromolecular therapeutics. Current in vitro BBB models fail to replicate this restrictive behavior due to poor integration of the endothelial cells with supporting cells (pericytes and astrocytes) following the correct anatomical organization observed in vivo. We report the coculture of mouse brain microvascular endothelial cells (b.End3), pericytes, with/without C8-D1A astrocytes in layered microfluidic channels forming three-dimensional (3D) bi- and triculture models of the BBB. The live/dead assay indicated high viability of all cultured cells up to 21 days. Trans-endothelial electrical resistance (TEER) values confirmed the formation of intact monolayers after 3 days in culture and showed statistically higher values for the triculture model compared to the single and biculture models. Screening the permeability of [(14)C]-mannitol and [(14)C]-urea showed the ability of bi- and triculture models to discriminate between different markers based on their size. Further, permeability of [(14)C]-mannitol across the triculture model after 18 days in culture matched its reported permeability across the BBB in vivo. Mathematical calculations also showed that the radius of the tight junctions pores (R) in the triculture model is similar to the reported diameter of the BBB in vivo. Finally, both the bi- and triculture models exhibited functional expression of the P-glycoprotein efflux pump, which increased with the increase in the number of days in culture. These results collectively indicate that the triculture model is a robust in vitro model of the BBB.
authors.affiliation: Department of Biomedical Engineering, University of Michigan , 1101 Beal Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Jack D
authors.initials: JD
authors.lastname: Wang
authors.affiliation: Department of Biomedical Engineering, University of Michigan , 1101 Beal Avenue, Ann Arbor, Michigan 48109, United States.|Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez Canal University , Ismailia 415-22, Egypt.
authors.forename: El-Sayed
authors.initials: el-S
authors.lastname: Khafagy
authors.affiliation: Department of Biomedical Engineering, University of Michigan , 1101 Beal Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Khalil
authors.initials: K
authors.lastname: Khanafer
authors.affiliation: Department of Biomedical Engineering, University of Michigan , 1101 Beal Avenue, Ann Arbor, Michigan 48109, United States.|Macromolecular Science and Engineering Program, University of Michigan , 2300 Hayward Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Takayama
authors.affiliation: Department of Biomedical Engineering, University of Michigan , 1101 Beal Avenue, Ann Arbor, Michigan 48109, United States.|Macromolecular Science and Engineering Program, University of Michigan , 2300 Hayward Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Mohamed E H
authors.initials: ME
authors.lastname: ElSayed
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000378:metabolism; D001812:Blood-Brain Barrier; D001921:Brain / Q000166:cytology* / Q000378:metabolism; D002199:Capillary Permeability; D018929:Cell Culture Techniques; D002463:Cell Membrane Permeability; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D044085:Microfluidics; D020286:Pericytes / Q000166:cytology* / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1
keywords: 3D in vitro models; C8-D1A astrocytes; b.End3 endothelial cells; blood−brain barrier; coculture systems; layered microfluidic channels