Low-density neuronal networks cultured using patterned poly-l-lysine on microelectrode arrays (original) (raw)
Related papers
Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference, 2005
Rectangular networks of rat hippocampal neurons have been produced on microelectrode arrays (MEAs). The crossing points of networks were located at the recording electrode sites by aligned microcontact printing (μCP) technique. Polydimethysiloxane (PDMS) stamp was fabricated to print fine poly-L-lysine (PLL) patterns of 2 -width lines for neurites and 20 -diameter circles for cell bodies. Different densities of neurons were applied on the PLL-stamped MEAs to find how a low density of neurons still has the functional connectivity. From the neural network applied with a density of 200 cells/mm2, we could observe signal propagation among spontaneous activities. Electrical responses were also evoked by 200 current pulse stimulation with 50 pulse width. Immunocytostaining was employed to identify dendrites, synapses, and nuclei in the patterned neurons.
Analysis of low-density neuronal networks on microcontact printed microelectrode arrays
Hippocampal neuronal networks were cultured on poly-L-lysine stamped microelectrode array. The neurons plated with low density enabled us to monitor electrical activities in a single cell level and to observe the signal propagation through the network. We found that electrically functional synapses still existed even in the density as low as 200 cells/mm 2. The cells were evoked by electrical current and high-K +. To identify what kinds of synapse are dominant in the cultured neurons, DNQX, AMPA antagonist were applied into the cultures. By scanning electron microscopy and immunocytostaining, the cultured neurons were visualized.
Patterning to enhance activity of cultured neuronal networks
IEE Proceedings - Nanobiotechnology, 2004
Embryonic rat hippocampal neurons were cultured in order to gain insights into how small networks of neurons interact. The principal observations are the electrical activities recorded with the electrode arrays, primarily action potentials both spontaneous and evoked. Several lithographic techniques were developed for controlling with micrometer precision the patterns of surface molecules in order to control neuronal attachment and growth. Cytophilic polylysine against protein repellent and hence cytophobic polyethylene glycol were used. By combining the cellular lithography with the microelectrode arrays it was possible to guide neurons preferentially to electrodes and to begin to investigate the question as to whether the geometric pattern of a neuronal network influences the patterns of its neuroelectric activity. It is clear that the techniques are adequate to ensure contact of neurons to electrodes but not to ensure the recording of signals, even when neurons lie directly on top of electrodes. The maturation of neuroelectric activity depends on the growth of glia within the culture, such that spontaneous activity appears to become robust when the number of glia is roughly the same as the number of neurons.
Multichannel recording and stimulation of neuronal cultures grown on microstamped poly-D-lysine
The 26th Annual International Conference of the IEEE Engineering in Medicine and Biology Society
We report progress toward designable, reproducible, patterned in vitro neuronal cultures. Cell adhesive proteins were directly microcontact printed on microelectrode arrays (MEAs) used as substrates for hippocampal neurons grown in a defined culture medium. The patterned neuronal network circuits were maintained up to one month. The recorded activity was comparable to that of cultures grown on unpatterned uniform surfaces. Time-locked evoked responses were recorded across the networks.
IEEE Transactions on Biomedical Engineering, 2000
We describe a method for producing high-resolution chemical patterns on surfaces to control the attachment and growth of cultured neurons. Microcontact printing has been extended to allow the printing of µm-scale protein lines aligned to an underlying pattern of planar microelectrodes. Poly-L-lysine (PL) lines have been printed on the electrode array for electrical studies on cultured neural networks. Rat hippocampal neurons showed a high degree of attachment selectivity to the PL and produced neurites that faithfully grew onto the electrode recording sites.
IEEE transactions on bio-medical engineering, 2007
Toward the development of biocompatible surfaces for implantable electrode arrays and the creation of patterned neuronal networks, the impact of select biochemical substrates [poly-D-lysine (PDL), polyornithine (PO), polyethylenimine (PEI), and a basement membrane extract (BM)] on network morphology and spontaneous electrophysiological activity of dissociated hippocampal neurons was investigated. Cultured in serum-free Neurobasal medium at 100 000 cells/cm(2), neurons attached to each substrate. PDL, PO, and PEI induced little or no neuronal clustering and process fasciculation, whereas the addition of BM promoted these features. The ratios of somas to processes, and axons to dendrites, as determined by immunohistochemical staining and image analysis were comparable across all substrates. Spontaneous firing was recorded using planar multielectrode arrays (MEAs) at the third week in vitro for the two most divergent morphologies according to Euclidian cluster analysis, namely those in...
Extracellular Recordings From Patterned Neuronal Networks Using Planar Microelectrode Arrays
IEEE Transactions on Biomedical Engineering, 2004
Neuronal cell networks have been reconstructed on planar microelectrode arrays (MEAs) from dissociated hippocampal pyramidal neurons. Microcontact printing ( CP) and a photoresist-liftoff method were used to selectively localize poly-L-lysine (PLL) on the surface of MEAs. Haptotaxis led to the organization of the neurons into networks localized adjacent to microelectrodes. Various grids of PLL with 2-25m-wide lines spaced by 50-200 m with 15-25-m nodes at intersection points were used to guide cell body attachment and neurite outgrowth. Bursting activity with spike amplitude attenuation was observed, and multichannel recordings detected instances of coincident firing activity. Finally, we present here an extracellular recording from a 2 m bundle of guided neurites.
Microelectrode Array Recordings of Patterned Hippocampal Neurons for Four Weeks
Biomedical Microdevices, 2000
Recent advances in cell biology and surface patterning make possible the construction of in vitro neural networks for long-term, multichannel recording studies. Towards this goal, we have demonstrated the recording of spontaneous electrical activity from rat embryonic hippocampal neurons confined to parallel lines which overlay the microelectrode array. The neurons adhered to adsorbed poly-D-lysine patterns and remained alive on the pattern for up to one month. Recordable, extracellular electrical activity began as early as 6 days in vitro and continued for the duration of the culture. Average amplitude of detected action potentials ranged between 70 μ V to 150 μ V measured from baseline to peak, consistent with results from unpatterned culture technologies.
Neuronal cell patterning on a multi-electrode array for a network analysis platform
Biomaterials, 2013
We studied neuronal cell patterning on a commercial multi-electrode array (MEA). We investigated the surface chemical modification of MEA in order to immobilize Poly-D-lysine (PDL) and then to pattern PDL with a photolithographic method using vacuum ultraviolet light (VUV). We have clarified that the PDL layer was not fully decomposed but was partially fragmented by short-time irradiation with VUV, resulting in a change in the cell adhesiveness of the PDL. We succeeded in patterning primary rat cortex cells without manipulating the cells on MEA more than two months. This cell-adhesiveness change induced by VUV can be applied to any immobilized PDL on other kinds of MEA and culturing substrate. We conducted electrophysiological measurements and found that the patterned neuronal cells were sufficiently matured and developed neural networks, demonstrating that our patterning method is useful for a neuronal network analysis platform.
Techniques for patterning and guidance of primary culture neurons on micro-electrode arrays
Sensors and Actuators B-chemical, 2002
In this work, two different micro-patterning methods for use in the growth guidance of dissociated primary culture neuron cells are compared: open chemically patterned growth substrates and enclosed micro-fluidic channels. Open, chemically patterned growth substrates are prepared by photolithographically patterning perfluoropolymer barrier structures on glass substrates. Neural pathways are created when poly-l-lysine is selectively adsorbed on the glass to form a cytophilic growth matrix. Adsorption of albumin proteins on the perfluoropolymer regions renders the surface cytophobic. In a second method, a three-dimensional micro-fluidic system, fabricated from PDMS is tested as a way to guide neural growth through total confinement and for cell placement. Both of these methods are tested for alignment and compatibility on a commercially available micro-electrode array (MED64). Biological culture and imaging techniques are considered.