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

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

doi:10.1186/s12987-015-0015-9

Abstract

BackgroundPrimary 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.MethodsPrimary 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.ResultsThe 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.ConclusionsThe data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

Highlights

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
Establishment of BBB in primary culture Three cell types were used to construct the three different types of in vitro BBB models (Fig. 1a). Based on their respective expression of the cell specific markers zona occludens 1 (ZO1), alpha-smooth muscle actin (α-SMA) and glial fibrillary acidic protein (GFAP), the three isolated cell types were identified as BCECs, pericytes and astrocytes, respectively (Fig. 1b)
transcellular endothelial electric resistance (TEER) measurements were not conducted beyond day 4, since TEER values decreased below 130 Ω cm2, at which stage in vitro BBB models using rat endothelial cells get insufficiently tight [36]

Summary

Introduction

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. The blood-brain barrier (BBB) denotes the interface between the circulation and the central nervous system (CNS) It is formed by non-fenestrated brain capillary endothelial cells (BCECs) that control the flux of substances into the CNS. Other non-neuronal cells of the CNS, mainly astrocytes and pericytes, form the neurovascular unit together with BCECs and support the function of the BBB to restrict both paracellular and transcellular transport pathways to the CNS [1,2,3,4]. These junctions lead to the formation of major gradients for inorganic and organic solutes between the brain and the blood plasma This accounts for the existence of high transcellular endothelial electric resistance (TEER) across BCECs [5]. The expression of several efflux transporter proteins like the ATP-binding cassette transporters prevents exogenous substances that are lipophilic in nature to pass through the BCECs [6,7,8]

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Conclusion