Joachim Wegener

A Nanogel for Ratiometric Fluorescent Sensing of Intracellular pH Values

A ratiometric fluorescent nanogel can sense pH over a range that is applicable to physiological studies. It can be easily prepared and made pH-responsive by addition of a pH probe and a FRET system that utilizes the gel to hold dyes in close proximity (see picture; overlay of coumarin dye and Nile Red fluorescence in kidney cells).

Cell adhesion monitoring using a quartz crystal microbalance: comparative analysis of different mammalian cell lines

Abstract The quartz crystal microbalance (QCM) has been widely accepted as a sensitive technique to follow adsorption processes in gas as well as in liquid environments. However, there are only a few reports about the use of this technique to monitor the attachment and spreading of mammalian cells onto a solid support in culture. Using a QCM-setup we investigated the time course of cell attachment and spreading as a function of seeding density for three widespread and frequently used cell lines (MDCK strains I and II and Swiss 3T3-fibroblasts). Results were found to be in good agreement with the geometrical properties of the individual cell types. The shifts of the resonance frequency associated with confluent cell layers on top of the quartz resonators were found to be dependent on the cell species [MDCK-I: (320±20) Hz; MDCK-II: (530±25) Hz; 3T3: (240±15) Hz] reflecting their individual influence on the shear oscillation of the resonator. These findings are discussed with respect to the basic models of materials in contact with an oscillating quartz resonator. We furthermore showed by inhibition-assays using soluble RGD-related peptides, that only specific, integrin mediated cell adhesion is detected using this QCM approach, whereas the sole presence of the cellular body in close vicinity to the resonator surface is barely detectable.

Ultra-Small, Highly Stable, and Sensitive Dual Nanosensors for Imaging Intracellular Oxygen and pH in Cytosol

We report on the first dual nanosensors for imaging of pH values and oxygen partial pressure in cells. The sensors have a unique nanostructure in that a soft core structure is rigidized with a silane reagent, while poly(ethylene glycol) chains form an outer shell. Lipophilic oxygen-sensitive probes and reference dyes are encapsulated inside the hydrophobic core, while a pH-sensitive probe is covalently attached to the poly(ethylene glycol) end-group on the shell. The core/shell structure renders the nanosensors well dispersed and highly stable in various kinds of aqueous media. Their average size is 12 nm, and they respond to both pH and oxygen in the physiological range. They do not pass cell membranes, but can be internalized into the cellular cytosol by electroporation, upon which they enable sensing and imaging of pH values and oxygen with high spatial resolution. The nanosensor strategy shown here is expected to be applicable to the development of various other kinds of multiple nanosensors for in vivo studies.

Impedance analysis of epithelial and endothelial cell monolayers cultured on gold surfaces

The present study describes a new method to determine transepithelial and transendothelial electrical resistances (TER) of cultured cell monolayers which is based on impedance analysis. To obtain impedance data of the epithelia or endothelia under investigation, we developed special measuring chambers that allow to culture the cells on gold surfaces that are used as measuring electrodes. Impedance analysis is carried out in the frequency range from 1 to 10(5) s-1 under normal culture conditions using a self-developed continuous wave impedance spectrometer. Evaluation of impedance data is achieved by fitting (NLSQ) the parameters of appropriate equivalent circuits to the experimental data. We investigated cell monolayers of primary cultured endothelial cells isolated from porcine brain microvessels, epithelial cells from porcine choroid plexus as well as those of the epithelial cell line MDCK. Transepithelial resistances were found to be in good agreement with published data. The main advantages of the new technique are the ability (i) to use multi-electrode arrays that allow to determine TERs at different locations of a given cell monolayer; (ii) to carry out impedance analysis under normal culture conditions; and (iii) to obtain TER values of cell monolayers grown on impermeable supports, which means that conditions cells are normally exposed to in ordinary culture dishes are maintained.

Tyrosine phosphatase inhibition induces loss of blood–brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.

Toxicity of gold-nanoparticles: Synergistic effects of shape and surface functionalization on micromotility of epithelial cells

Abstract Nanoparticle exposure is monitored by a combination of two label-free and non-invasive biosensor devices which detect cellular shape and viscoelasticity (quartz crystal microbalance), cell motility and the dynamics of epithelial cell-cell contacts (electric cell-substrate impedance sensing). With these tools we have studied the impact of nanoparticle shape on cellular physiology. Gold (Au) nanoparticles coated with CTAB were synthesized and studied in two distinct shapes: Spheres with a diameter of (43 ± 4) nm and rods with a size of (38 ± 7) nm × (17 ± 3) nm. Dose-response experiments were accompanied by conventional cytotoxicity tests as well as fluorescence and dark-field microscopy to visualize the intracellular particle distribution. We found that spherical gold nanoparticles with identical surface functionalization are generally more toxic and more efficiently ingested than rod-shaped particles. We largely attribute the higher toxicity of CTAB-coated spheres as compared to rod-shaped particles to a higher release of toxic CTAB upon intracellular aggregation.

Use of electrochemical impedance measurements to monitor beta-adrenergic stimulation of bovine aortic endothelial cells.

Abstract Due to the high permeability of endothelial cell layers derived from macrovascular vessels, precise determination of their barrier function towards ion movement requires refined experimental techniques. We thus cultured bovine aortic endothelial cells (BAEC) directly on thin gold-film electrodes and measured the electrochemical impedance to study their passive electrical properties in general and during β-adrenergic stimulation. Impedance spectra (10–2·106 Hz) of confluent cell monolayers revealed that the electrical characteristics of the cells can be modelled by a simple resistor-capacitor parallel network. Under control conditions the overall resistance of confluent BAEC monolayers was 3.6±0.6 Ω·cm2 (n=30) and the capacitance was 0.6±0.1 µF/cm2. Both quantities are discussed with respect to morphological characteristics of these cells. Stimulation of BAECs with the synthetic β-adrenoceptor agonist isoproterenol leads to a concentration-dependent, highly specific increase of the cell layer resistance characterized by a concentration for half-maximal response (EC50) of 0.3±0.1 µM. The cell layer capacitance, however, remained unaffected. Using impedance measurements at a single frequency, we analysed the response of BAECs to treatment with isoproterenol in comparison with several chemically unrelated compounds known to stimulate the adenosine 3’,5’-cyclic monophosphate (cAMP)-dependent signal transduction cascade. These studies confirmed that the enhancement of the cell layer resistance after β-adrenergic stimulation is mediated by an increase in intracellular cAMP.

The quartz crystal microbalance as a novel means to study cell-substrate interactions In situ

The quartz crystal microbalance (QCM) was first introduced as a mass sensor in gas phase and in vacuum. Since oscillator circuits capable of exciting shear vibrations of quartz resonators under liquid loading have been developed, the QCM became accepted as a new, powerful technique to follow adsorption processes at solid-liquid interfaces in chemical and biological research. Lately, the QCM technique has attracted considerable interest as a novel means to monitor cell-substrate interactions of mammalian cells in vitro. Because the establishment and modulation of cell-substrate contacts is important for many physiological processes, and potent techniques to measure these interactions noninvasively are rare, the present review highlights applications of the QCM technique in this field. The suitability of the QCM device to monitor attachment and spreading of mammalian cells in real time has been well established. The QCM response is dependent on the individual cell type that is examined. In order to identify the sources for these cell-type-specific results of QCM readings, and to understand the information content of the signal, attempts have been made to decompose the overall QCM response into subcellular contributions. The aforementioned subjects, together with a condensed introduction into the QCM technology, are included in this article.

Self-referenced RGB colour imaging of intracellular oxygen

Intracellular oxygen is an important indicator for cell metabolism and respiration. We have designed self-referenced RGB PEBBLEs that enable a simple readout of the intracellular oxygen distribution with conventional wide-field microscopy and a standard RGB digital camera. The RGB PEBBLEs consist of a hydrophobic matrix covered with amino groups on the surface to confer water-dispersibility. Two luminophores are incorporated in a hydrophobic polystyrene matrix that is highly permeable to oxygen. In polystyrene, the dyes are largely protected from quenching or aggregation by cellular components. The dyes have been selected to match the green and the red channel of digital cameras. While the red emission of the oxygen probe is highly sensitive to oxygen with a quenching response of 74%, the green emission of the reference dye is stable under varying oxygen concentrations. Ratiometric images of intracellular oxygen have been acquired that are inherently resistant to fluctuations in absolute signal intensities. As RGB PEBBLEs respond within seconds to changing oxygen concentrations, they are amenable to monitoring fast cellular dynamics.

Barrier function of porcine choroid plexus epithelial cells is modulated by cAMP-dependent pathways in vitro.

In the present paper, we demonstrate that the barrier properties of primary cultured epithelial cells isolated from porcine choroid plexus are regulated by cAMP-dependent signal transduction pathways in vitro. Triggering cAMP-connected cascades in cell layers grown on permeable filters with cAMP-analogues or forskolin led to a significant increase of transepithelial electrical resistances and a pronounced reduction in the permeation rate of a 4 kDa-dextran probe. In dose-response experiments using the cAMP-analogue 8-(4-chlorophenylthio)-cAMP transepithelial electrical resistances were observed to increase above a threshold concentration ranging between 10(-5.5) and 10(-5) M. Additional impedance studies performed with confluent cell layers grown on gold-film electrodes revealed that the observed changes in transepithelial resistances and presumably also in macromolecular permeation rates were not entirely caused by a reinforcement of intercellular junctions but also contained contributions from changes in the cell-substrate adhesion pattern. These inherent contributions to the electrical resistance and macromolecular permeability are caused by a restricted diffusion pathway between basal plasma membrane and culture substrate that have to be considered in data analysis, especially when leaky cell layers on filter substrates with low pore densities are used.