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Fluids and Barriers of the CNS
Published in: Fluids and Barriers of the CNS 1/2015

Open Access 01-12-2015 | Research

Transfection of brain capillary endothelial cells in primary culture with defined blood–brain barrier properties

Authors: Annette Burkhart, Louiza Bohn Thomsen, Maj Schneider Thomsen, Jacek Lichota, Csilla Fazakas, István Krizbai, Torben Moos

Published in: Fluids and Barriers of the CNS | Issue 1/2015

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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.
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Metadata
Title
Transfection of brain capillary endothelial cells in primary culture with defined blood–brain barrier properties
Authors
Annette Burkhart
Louiza Bohn Thomsen
Maj Schneider Thomsen
Jacek Lichota
Csilla Fazakas
István Krizbai
Torben Moos
Publication date
01-12-2015
Publisher
BioMed Central
Published in
Fluids and Barriers of the CNS / Issue 1/2015
Electronic ISSN: 2045-8118
DOI
https://doi.org/10.1186/s12987-015-0015-9

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