title: Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.
abstract: BACKGROUND
  Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity.
  
  
  METHODS
  Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation.
  
  
  RESULTS
  The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer.
  
  
  CONCLUSIONS
  The data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.
authors.affiliation: Department of Health Science and Technology, Laboratory of Neurobiology, Biomedicine Group, Aalborg University, Frederik Bajers Vej 3B, 1.216, 9220, Aalborg East, Denmark. abl@hst.aau.dk.
authors.forename: Annette
authors.initials: A
authors.lastname: Burkhart
authors.affiliation: Department of Health Science and Technology, Laboratory of Neurobiology, Biomedicine Group, Aalborg University, Frederik Bajers Vej 3B, 1.216, 9220, Aalborg East, Denmark. lbt@hst.aau.dk.
authors.forename: Louiza Bohn
authors.initials: LB
authors.lastname: Thomsen
authors.affiliation: Department of Health Science and Technology, Laboratory of Neurobiology, Biomedicine Group, Aalborg University, Frederik Bajers Vej 3B, 1.216, 9220, Aalborg East, Denmark. mst@hst.aau.dk.
authors.forename: Maj Schneider
authors.initials: MS
authors.lastname: Thomsen
authors.affiliation: Department of Health Science and Technology, Laboratory of Neurobiology, Biomedicine Group, Aalborg University, Frederik Bajers Vej 3B, 1.216, 9220, Aalborg East, Denmark. jlichota@hst.aau.dk.
authors.forename: Jacek
authors.initials: J
authors.lastname: Lichota
authors.affiliation: Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary. fazakas.csilla@brc.mta.hu.
authors.forename: Csilla
authors.initials: C
authors.lastname: Fazakas
authors.affiliation: Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary. krizbai.istvan@brc.mta.hu.
authors.forename: István
authors.initials: I
authors.lastname: Krizbai
authors.affiliation: Department of Health Science and Technology, Laboratory of Neurobiology, Biomedicine Group, Aalborg University, Frederik Bajers Vej 3B, 1.216, 9220, Aalborg East, Denmark. tmoos@hst.aau.dk.
authors.forename: Torben
authors.initials: T
authors.lastname: Moos
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000502:physiology; D001812:Blood-Brain Barrier / Q000166:cytology / Q000378:metabolism*; D002199:Capillary Permeability; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002478:Cells, Cultured; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D020286:Pericytes / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection