Helmut Franke

Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood–brain barrier in vitro

Studies on drug entry into the brain and permeation of the blood-brain barrier start to gain more and more importance in neuropharmaceutical research in order to develop new drugs for the therapy of central nervous system diseases. Procedures that provide quick access to permeation properties of those drugs with high throughput are difficult to achieve with animal models. Although various useful cell culture models approaching this issue have been described, results are often not comparable among each other unless determined with an equal experimental setup. Reproducibility of cell culture methods as well as corresponding findings gathered with these tools are often impeded due to the lack of details in experimental manuals. Here we present a precise manual for preparation and maintenance of porcine brain microvessel endothelial cells, serving as a culture model of the blood-brain barrier. Furthermore experimental details for blood-brain barrier transport investigations are presented. Validation of this model was carried out by determination of bioelectric properties and permeation experiments using various marker molecules reflecting paracellular and transcellular blood-brain barrier penetration. Results obtained with our model are closely resembling the in vivo-situation although astrocytes are not included. This simplification of the system is one of the major advantages towards robot derived cell cultures necessary for high throughput screening.

An improved low-permeability in vitro-model of the blood–brain barrier: transport studies on retinoids, sucrose, haloperidol, caffeine and mannitol

Primary cultures of porcine brain capillary endothelial cells grown on collagen coated polycarbonate membranes were used to build up an in vitro-model for the blood-brain barrier. Improved cultivation techniques allowed cell-storage and experiments under serum-free conditions. We employed this model to perform permeability studies in vitro with the radioactively labelled marker substances sucrose, retinoic acid, retinol, haloperidol, caffeine, and mannitol. Permeability values obtained with this blood-brain barrier model (1. 0x10-6 cm/s for sucrose, 6.2x10-6 cm/s for retinoic acid, 4.8x10-6 cm/s for retinol, 49.5x10-6 cm/s for haloperidol, 62.4x10-6 cm/s for caffeine, and 1.8x10-6 cm/s for mannitol) show a good correlation to data which are already known from in vivo-experiments. As judged by the sucrose permeability our blood-brain barrier model is less permeable than numerous other models published so far. Therefore it represents a powerful tool for in vitro-prediction of blood-brain barrier permeability of drugs and offers the possibility to scan a large quantity of drugs for their potential to enter the brain.

Effects of universal mobile telecommunications system (UMTS) electromagnetic fields on the blood-brain barrier in vitro

Abstract Franke, H., Streckert, J., Bitz, A., Goeke, J., Hansen, V., Ringelstein, E. B., Nattkämper, H., Galla, H-J. and Stögbauer, F. Effects of Universal Mobile Telecommunications System (UMTS) Electromagnetic Fields on the Blood-Brain Barrier In Vitro. Radiat. Res. 164, 258–269 (2005). The extensive use of mobile phone communication has raised public concerns about adverse health effects of radiofrequency (RF) electromagnetic fields (EMFs) in recent years. A central issue in this discussion is the question whether EMFs enhance the permeability of the blood-brain barrier (BBB). Here we report an investigation on the influence of a generic UMTS (Universal Mobile Telecommunications System) signal on barrier tightness, transport processes and the morphology of porcine brain microvascular endothelial cell cultures (PBEC) serving as an in vitro model of the BBB. An exposure device with integrated online monitoring system was developed for simultaneous exposure and measuring of transendothelial electrical resistance (TEER) to determine the tightness of the BBB. PBEC were exposed continuously for up to 84 h at an average electric-field strength of 3.4–34 V/m (maximum 1.8 W/kg) ensuring athermal conditions. We did not find any evidence of RF-field-induced disturbance of the function of the BBB. After and during exposure, the tightness of the BBB quantified by 14C-sucrose and serum albumin permeation as well as by TEER remained unchanged compared to sham-exposed cultures. Permeation of transporter substrates at the BBB as well as the localization and integrity of the tight-junction proteins occludin and ZO1 were not affected either.

Electrical resistance measurements on cerebral capillary endothelial cells — a new technique to study small surface areas

We present a new method which allows resistance measurements in selected cell monolayer areas with a size of less than one mm2. Up to now cell-covered macroscopic filters with areas up to 5 cm2 have been used giving reliable results only in the absence of inhomogeneities or contaminating cells. The new measuring device is posed on the microscope optic to allow an optical characterization of the measuring area and enables us to scan the cell-covered filter. This method has been applied to determine a reversible modulation of the tightness of intercellular contacts between epithelial or endothelial cells in culture. Tight junction resistance is modulated by Ca2+ and basic amino acids in cultured porcine cerebral microvascular endothelial cells that represent the blood-brain barrier.

Five years later: The current status of the use of proteomics and transcriptomics in EMF research

The World Health Organizations and Radiation and Nuclear Safety Authoritys “Workshop on Application of Proteomics and Transcriptomics in Electromagnetic Fields Research” was held in Helsinki in the October/November 2005. As a consequence of this meeting, Proteomics journal published in 2006 a special issue “Application of Proteomics and Transcriptomics in EMF Research” (Vol. 6 No. 17; Guest Editor: D. Leszczynski). This Proteomics issue presented the status of research, of the effects of electromagnetic fields (EMF) using proteomics and transcriptomics methods, present in 2005. The current overview/opinion article presents the status of research in this area by reviewing all studies that were published by the end of 2010. The review work was a part of the European Cooperation in the Field of Scientific and Technical Research (COST) Action BM0704 that created a structure in which researchers in the field of EMF and health shared knowledge and information. The review was prepared by the members of the COST Action BM0704 task group on the high‐throughput screening techniques and electromagnetic fields (TG‐HTST‐EMF).

New Techniques to Study Transepithelial and Transendothelial Resistances of Cultured Cells

Measurement of transepithelial electrical resistances (TER) is a very common tool for investigations concerning epithelial and endothelial barrier properties. We here introduce two new and powerful techniques for the determination of TERs that might open further applications. The first method takes advantage of the wide spread DC technique and allows to study individual colonies of a cell monolayer grown on waterpermeable filter supports. The second technique is based on AC impedance analysis and enables to determine TERs of cell monolayers grown on gold surfaces.