Dielectrophoretic trapping of dissociated fetal cortical rat neurons

Recording and stimulating neuronal activity at multiple sites can be realized with planar microelectrode arrays. Efficient use of such arrays requires each site to be covered by at least one neuron. By application of dielectrophoresis (DEP), neurons can be trapped onto these sites. This study investigates negative dielectrophoretic trapping of fetal cortical rat neurons. A planar quadrupole microelectrode structure was used for the creation of a nonuniform electric field. The field was varied in amplitude (1, 3, and 5 V) and frequency (10 kHz-50 MHz). Experimental results were compared with a theoretical model to investigate the yield (the number of neurons trapped in the center of the electrode structure) with respect to time, amplitude and frequency of the field. The yield was a function of time1/3 according to theory. However, unlike the model predicted, an amplitude-dependent frequency behavior was present and unexpected peaks occurred in the DEP-spectra above 1 MHz. Gain/phase measurements showed a rather unpredictable behavior of the electrode plate above 1 MHz, and temperature measurement showed that heating of the medium influenced the trapping effect, especially for larger amplitudes and higher frequencie

Neuroelectronic interfacing with cultured multielectrode arrays toward a cultured probe

Efficient and selective electrical stimulation and recording of neural activity in peripheral, spinal, or central pathways requires multielectrode arrays at micrometer scale. ¿Cultured probe¿ devices are being developed, i.e., cell-cultured planar multielectrode arrays (MEAs). They may enhance efficiency and selectivity because neural cells have been grown over and around each electrode site as electrode-specific local networks. If, after implantation, collateral sprouts branch from a motor fiber (ventral horn area) and if they can be guided and contacted to each ¿host¿ network, a very selective and efficient interface will result. Four basic aspects of the design and development of a cultured probe, coated with rat cortical or dorsal root ganglion neurons, are described. First, the importance of optimization of the cell-electrode contact is presented. It turns out that impedance spectroscopy, and detailed modeling of the electrode-cell interface, is a very helpful technique, which shows whether a cell is covering an electrode and how strong the sealing is. Second, the dielectrophoretic trapping method directs cells efficiently to desired spots on the substrate, and cells remain viable after the treatment. The number of cells trapped is dependent on the electric field parameters and the occurrence of a secondary force, a fluid flow (as a result of field-induced heating). It was found that the viability of trapped cortical cells was not influenced by the electric field. Third, cells must adhere to the surface of the substrate and form networks, which are locally confined, to one electrode site. For that, chemical modification of the substrate and electrode areas with various coatings, such as polyethyleneimine (PEI) and fluorocarbon monolayers promotes or inhibits adhesion of cells. Finally, it is shown how PEI patterning, by a stamping technique, successfully guides outgrowth of collaterals from a neonatal rat lumbar spinal cord explant, after six days in cultur

Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers

Investigating membrane breakdown of neuronal cells exposed to nonuniform electric fields by finite-element modeling and experiments

High electric field strengths may induce high cell membrane potentials. At a certain breakdown level the membrane potential becomes constant due to the transition from an insulating state into a high conductivity and high permeability state. Pores are thought to be created through which molecules may be transported into and out of the cell interior. Membrane rupture may follow due to the expansion of pores or the creation of many small pores across a certain part of the membrane surface. In nonuniform electric fields, it is difficult to predict the electroporated membrane area. Therefore, in this study the induced membrane potential and the membrane area where this potential exceeds the breakdown level is investigated by finite-element modeling. Results from experiments in which the collapse of neuronal cells was detected were combined with the computed field strengths in order to investigate membrane breakdown and membrane rupture. It was found that in nonuniform fields membrane rupture is position dependent, especially at higher breakdown levels. This indicates that the size of the membrane site that is affected by electroporation determines rupture

Electrokinetic confinement of axonal growth for dynamically configurable neural networks

Axons in the developing nervous system are directed via guidance cues, whose expression varies both spatially and temporally, to create functional neural circuits. Existing methods to create patterns of neural connectivity in vitro use only static geometries, and are unable to dynamically alter the guidance cues imparted on the cells. We introduce the use of AC electrokinetics to dynamically control axonal growth in cultured rat hippocampal neurons. We find that the application of modest voltages at frequencies on the order of 10[superscript 5] Hz can cause developing axons to be stopped adjacent to the electrodes while axons away from the electric fields exhibit uninhibited growth. By switching electrodes on or off, we can reversibly inhibit or permit axon passage across the electrodes. Our models suggest that dielectrophoresis is the causative AC electrokinetic effect. We make use of our dynamic control over axon elongation to create an axon-diode via an axon-lock system that consists of a pair of electrode ‘gates’ that either permit or prevent axons from passing through. Finally, we developed a neural circuit consisting of three populations of neurons, separated by three axon-locks to demonstrate the assembly of a functional, engineered neural network. Action potential recordings demonstrate that the AC electrokinetic effect does not harm axons, and Ca[superscript 2+] imaging demonstrated the unidirectional nature of the synaptic connections. AC electrokinetic confinement of axonal growth has potential for creating configurable, directional neural networks.National Institutes of Health (U.S.) (R01 EUREKA Award R01-NS066352

Localized Dielectric Loss Heating in Dielectrophoresis Devices

Temperature increases during dielectrophoresis (DEP) can affect the response of biological entities, and ignoring the effect can result in misleading analysis. The heating mechanism of a DEP device is typically considered to be the result of Joule heating and is overlooked without an appropriate analysis. Our experiment and analysis indicate that the heating mechanism is due to the dielectric loss (Debye relaxation). A temperature increase between interdigitated electrodes (IDEs) has been measured with an integrated micro temperature sensor between IDEs to be as high as 70 °C at 1.5 MHz with a 30 Vpp applied voltage to our ultra-low thermal mass DEP device. Analytical and numerical analysis of the power dissipation due to the dielectric loss are in good agreement with the experiment data

Creation of Defined Single Cell Resolution Neuronal Circuits on Microelectrode Arrays

The way cell-cell organization of neuronal networks influences activity and facilitates function is not well understood. Microelectrode arrays (MEAs) and advancing cell patterning technologies have enabled access to and control of in vitro neuronal networks spawning much new research in neuroscience and neuroengineering. We propose that small, simple networks of neurons with defined circuitry may serve as valuable research models where every connection can be analyzed, controlled and manipulated. Towards the goal of creating such neuronal networks we have applied microfabricated elastomeric membranes, surface modification and our unique laser cell patterning system to create defined neuronal circuits with single-cell precision on MEAs. Definition of synaptic connectivity was imposed by the 3D physical constraints of polydimethylsiloxane elastomeric membranes. The membranes had 20μm clear-through holes and 2-3μm deep channels which when applied to the surface of the MEA formed microwells to confine neurons to electrodes connected via shallow tunnels to direct neurite outgrowth. Tapering and turning of channels was used to influence neurite polarity. Biocompatibility of the membranes was increased by vacuum baking, oligomer extraction, and autoclaving. Membranes were bound to the MEA by oxygen plasma treatment and heated pressure. The MEA/membrane surface was treated with oxygen plasma, poly-D-lysine and laminin to improve neuron attachment, survival and neurite outgrowth. Prior to cell patterning the outer edge of culture area was seeded with 5x105 cells per cm and incubated for 2 days. Single embryonic day 7 chick forebrain neurons were then patterned into the microwells and onto the electrodes using our laser cell patterning system. Patterned neurons successfully attached to and were confined to the electrodes. Neurites extended through the interconnecting channels and connected with adjacent neurons. These results demonstrate that neuronal circuits can be created with clearly defined circuitry and a one-to-one neuron-electrode ratio. The techniques and processes described here may be used in future research to create defined neuronal circuits to model in vivo circuits and study neuronal network processing

Miniaturized bioanalytics to probe the function of membrane proteins

G-protein coupled receptors (GPCRs) are the most abundant class of proteins in the cell body. Such receptors are of major interest as potential therapeutic targets. Downscaling and parallelization of bioanalytics opens novel routes to rapidly screen and identify potential drugs with a decrease in regard to the costs, and elucidate novel functions of signaling networks under physiological conditions. Native vesicles are small autonomous biological containers, which are efficiently produced from all cell lines. They are composed of their mother cell plasma membrane and enclose part of their cytoplasm. Membrane receptors are then exposed at their surface and already demonstrate to induce cellular signaling when exposed to receptor ligands. Native vesicles were investigated in this present work, as novel possibilities to downscale receptor investigation in live cells, using neurokinin 1 receptor (NK1R) as a representative model. Here native vesicle production and purification was optimized. The influence of the cell cycle on the production efficiency was demonstrated. Biological proteins were downregulated in order to produce blebbing cells. Native vesicle characterization was achieved. The receptors also demonstrate to be efficiently labeled by agonist and antagonist, allowing to access information about the binding kinetics as well as KD values. The results are in agreement with those obtained in live cells. Native vesicles also demonstrate to internalize agonist after application, demonstrating receptor desensitization and signaling performance similar as live cells. Confocal microscopy shows that cells expressing the NK1R-CFP have two binding affinities for their main agonist, substance P. Similar results could be observed with flow cytometry. The high affinity binding is related to cholesterol content in the cell membrane and was abolished by cholesterol depletion with methyl-β-cyclodextrin. Micro-contact printing (µCP) was used to (bio)functionalize surfaces with proteins, polymers or functionalized nanoparticles. Precise sample positioning by micro-contact printing shows improves nuclear magnetic resonance excitation and detection, when performed with a planar microcoil probe. µCP was used to produce native vesicle arrays by two procedures, and fluorescent binding assay shows the binding of fluorescent ligands to the receptor. Laser tweezers allow manipulating cell membranes without requiring the use of polystyrene beads. From pulled membranes, native vesicles were produced. In addition from pulled membranes, large tethers were produced and artificial connections were established with neighboring cells. Intercellular communication was investigated by whole-cell patch clamp in dissociated primary dorsal root ganglion neurons after optical induced connection, as well as in HEK cells expressing Cx36. The lab-on-chip assay development demonstrates: the high production of native vesicles in microchannels; the efficient purification obtained by negative dielectrophoresis depletion in MEMS chips; perfect trapping and thus immobilization of native vesicles in a new optical multi-tweezer array. Fluorescence labeling was performed with native vesicles trapped in a multi-tweezer array inside microfluidic channel in the presence of two laminar flows. Optical multi-tweezer array setup shows to be the fastest and more efficient technique in order to perform immobilization and labeling of native vesicles in the microfluidic channel. It is presently the only technique to perform fluorescence measurements when maintaining objects trapped