Long-term in vivo impedance changes of subretinal microelectrodes implanted in dystrophic P23H rats.

Retinal prostheses are being developed to restore vision in blind patients with photoreceptor degeneration. Electrodes arrays were subretinally implanted in transgenic P23H rats with their photoreceptors degenerated. Electrical stability of the implants was evaluated by long-term monitoring of their impedance changes. Electrode impedances were found to increase by two log units over a three weeks period whereas no impedance increase was noted when the implants were located in the vitreous. In case of hemorrhage or major fibrous reactions, the impedance continued to increase steadily. After explantation, it recovered its initial value indicating no deterioration of the implant. Although the glial cell layer at the surface of the subretinal space was slightly larger, no major glial reaction was seen in direct contact to the implant. These results indicate that no functional testing should be considered before at least three weeks post implantation.

Surface protein patterns govern morphology, proliferation, and expression of cellular markers but have no effect on physiological properties of cortical precursor cells.

The ability to differentiate and give rise to neurons, astrocytes, and oligodendrocytes is an inherent feature of neural stem cells, which raises hopes for cell-based therapies of neurodegenerative diseases. However, there are many hurdles to cross before such regimens can be applied clinically. A considerable challenge is to elucidate the factors that contribute to neural differentiation. In this study, we evaluated the possibility of steering neuronal maturation by growing cortical precursor cells on microscale surface patterns of extracellular matrix (ECM) proteins. When the cells were encouraged to extend processes along lines of ECM proteins, they displayed a much more mature morphology, less proliferation capacity, and greater expression of a neuronal marker in comparison with cells grown in clusters on ECM dots. This implied that the growth pattern alone could play a crucial role for neural differentiation. However, in spite of the strikingly different morphology, when performing whole-cell patch-clamp experiments, we never observed any differences in the functional properties between cells grown on the two patterns. These results clearly demonstrate that morphological appearances are not representative measures of the functional phenotype or grade of neuronal maturation, stressing the importance of complementary electrophysiological evidence. To develop successful transplantation therapies, increased cell survival is critical. Because process-bearing neurons are sensitive and break easily, it would be of clinical interest to explore further the differentiating capacity of the cells cultured on the ECM dot pattern, described in this article, which are devoid of processes but display the same functional properties as neurons with mature morphology.

Cell culture imaging using microimpedance tomography.

We present a novel, inexpensive, and fast microimpedance tomography system for two-dimensional imaging of cell and tissue cultures. The system is based on four-electrode measurements using 16 planar microelectrodes (5 microm x 4 mm) integrated into a culture chamber. An Agilent 4294A impedance analyzer combined with a front-end amplifier is used for the impedance measurements. Two-dimensional images are obtained using a reconstruction algorithm. This system is capable of accurately resolving the shape and position of a human hair, yielding vertical cross sections of the object. Human epithelial stem cells (YF 29) are also grown directly on the device surface. Tissue growth can be followed over several days. A rapid resistivity decrease caused by permeabilized cell membranes is also monitored, suggesting that this technique can be used in electroporation studies.

Characterization and optimization of liquid electrodes for lateral dielectrophoresis.

Using the concept of insulator-based "electrodeless" dielectrophoresis, we present a novel geometry for shaping electric fields to achieve lateral deviation of particles in liquid flows. The field is generated by lateral planar metal electrodes and is guided along access channels to the active area in the main channel. The equipotential surfaces at the apertures of the access channels behave as vertical "liquid" electrodes injecting the current into the main channel. The field between a pair of adjacent liquid electrodes generates the lateral dielectrophoretic force necessary for particle manipulation. We use this force for high-speed deviation of particles. By adding a second pair of liquid electrodes, we focus a particle stream. The position of the focused stream can be swept across the channel by adjusting the ratio of the voltages applied to the two pairs. Based on conformal mapping, we provide an analytical model for estimating the potential at the liquid electrodes and the field distribution in the main channel. We show that the simulated particle trajectories agree with observations. Finally, we show that the model can be used to optimize the device geometry in different applications.

Bipolar resistivity profiling of 3D tissue culture.

We describe a new low-cost technique for continuous monitoring of the thickness of biofilms and tissue cultures, and we demonstrate the advantage of using electrodes of different dimensions to probe different depths of a sample. We have used electric impedance spectroscopy to monitor keratinocyte stem cells (YF29) growing on an array of Ti/Pt coplanar microelectrodes. The thickness of the sample was reconstructed by fitting the measurements to theoretical curves. We have developed an algorithm for the rapid calculation of the resistance through a multilayered sample. This algorithm is based on conformal mapping and the serial partial capacitance technique. The validity of the technique was tested by measuring the sedimentation rate of an alumina powder. Sample thicknesses between 10 and 80 microm could be measured with a resolution of a few microns using the device.

Two-dimensional impedance imaging of cell migration and epithelial stratification.

We present a miniaturized impedance imaging system, developed for 2D imaging of cell and tissue culture. The system is based on 16 microelectrodes (5 microm x 4 mm). An equivalent circuit for four-point (tetrapolar) impedance spectra was developed and validated. The system uses an Agilent 4294A impedance analyser combined with a front-end amplifier for the impedance measurements. Human epithelial stem cells (YF 29) were grown on the device surface. Cell migration speeds of 300 nm min(-1) following a "scratch" wound closure assay could be established. Using a commercial software developed for geophysical prospecting, we could generate impedance tomography images at 10 kHz revealing cell migration, increase of epithelial thickness and changes in tissue resistivity over a time course of several days.

Resistivity probing of multi-layered tissue phantoms using microelectrodes.

We present the use of an array of rectangular microelectrodes to discriminate between different resistivities in a thin, layered sample. Each electrode was 8 mm long and 200 nm thick. The electrode widths ranged from 20 to 500 microm. The electrodes were designed such that all pairs of consecutive electrodes had the same relative geometry, and therefore identical cell constants. A hydrogel-based tissue phantom, made by photopolymerization of 2-hydroxyethyl methacrylate (HEMA), was developed. By changing the hydrogel composition and the ionic strength of the storage medium, the resistivity of the hydrogels could be tuned between 100 omegam and 100 komegam. Using bipolar measurements, the tissue phantoms were characterized in the frequency range from 100 Hz to 30 MHz. The relative resistivity distribution of a three-layered structure composed of 120 microm sheets could be calculated and was shown to agree to within 7% of the bulk measurements. Potential clinical applications for this technique include probing of epithelial tissue and skin cancer screening.

Formation of geometrically complex lipid nanotube-vesicle networks of higher-order topologies.

We present a microelectrofusion method for construction of fluid-state lipid bilayer networks of high geometrical complexity up to fully connected networks with genus = 3 topology. Within networks, self-organizing branching nanotube architectures could be produced where intersections spontaneously arrange themselves into three-way junctions with an angle of 120 degrees between each nanotube. Formation of branching nanotube networks appears to follow a minimum-bending energy algorithm that solves for pathway minimization. It is also demonstrated that materials can be injected into specific containers within a network by nanotube-mediated transport of satellite vesicles having defined contents. Using a combination of microelectrofusion, spontaneous nanotube pattern formation, and satellite-vesicle injection, complex networks of containers and nanotubes can be produced for a range of applications in, for example, nanofluidics and artificial cell design. In addition, this electrofusion method allows integration of biological cells into lipid nanotube-vesicle networks.