title: An in vitro blood-brain barrier model combining shear stress and endothelial cell/astrocyte co-culture.
abstract: BACKGROUND
  In vitro blood-brain barrier (BBB) models can be useful for understanding leukocyte-endothelial interactions at this unique vascular-tissue interface. Desirable features of such a model include shear stress, non-transformed cells and co-cultures of brain microvascular endothelial cells with astrocytes. Recovery of transmigrated leukocytes for further analysis is also appealing.
  
  
  METHODS
  We report an in vitro BBB model for leukocyte transmigration incorporating shear stress with co-culture of conditionally immortalized human endothelial cell line (hBMVEC) and human astrocyte cell line (hAST). Transmigrated leukocytes can be recovered for comparison with input and non-transmigrated cells.
  
  
  RESULTS
  hBMVEC and hAST exhibited physiological and morphological BBB properties when cocultured back-to-back on membranes. In particular, astrocyte processes protruded through 3 μm membrane pores, terminating in close proximity to the hBMVEC with a morphology reminiscent of end-feet. Co-culture with hAST also decreased the permeability of hBMVEC. In our model, astrocytes promoted transendothelial leukocyte transmigration.
  
  
  METHODS
  This model offers the opportunity to evaluate whether BBB properties and leukocyte transmigration across cytokine-activated hBMVEC are influenced by human astrocytes.
  
  
  CONCLUSIONS
  We present a BBB model for leukocyte transmigration incorporating shear stress with co-culture of hBMVEC and hAST. We demonstrate that hAST promoted leukocyte transmigration and also increased certain barrier functions of hBMVEC. This model provides reproducible assays for leukocyte transmigration with robust results, which will enable further defining the relationships among leukocytes and the cellular elements of the BBB.
authors.affiliation: Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, United States.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Takeshita
authors.affiliation: Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, United States.
authors.forename: Birgit
authors.initials: B
authors.lastname: Obermeier
authors.affiliation: Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, United States.
authors.forename: Anne
authors.initials: A
authors.lastname: Cotleur
authors.affiliation: Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Sano
authors.affiliation: Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kanda
authors.affiliation: Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, United States. Electronic address: ransohr@ccf.org.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Ransohoff
mesh_terms: D000328:Adult; D001253:Astrocytes / Q000502:physiology*; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000502:physiology* / Q000648:ultrastructure; D002478:Cells, Cultured; D062507:Claudin-5 / Q000378:metabolism; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000502:physiology* / Q000648:ultrastructure; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D008297:Male; D055806:Microvessels / Q000166:cytology; D008875:Middle Aged; D008954:Models, Biological; D062793:Occludin / Q000378:metabolism; D013314:Stress, Mechanical; D013696:Temperature; D057705:Transendothelial and Transepithelial Migration; D055815:Young Adult; D062826:Zonula Occludens-1 Protein / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D062507:Claudin-5; D005904:Glial Fibrillary Acidic Protein; D062793:Occludin; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D014841:von Willebrand Factor
keywords: Blood–brain barrier; Co-culture; Human astrocyte cell line; Human endothelial cell line; In vitro model; Leukocyte transmigration; Migration assay; Shear forces