This work demonstrates the design, fabrication, packaging, characterization, and functionality of an electrically and fluidically active three-dimensional micro-electrode array (3D MEA) for use with neuronal cell cultures. The successful function of the device implies that this basic concept-construction of a 3D array with a layered approach-can be utilized as the basis for a new family of neural electrode arrays. The 3D MEA prototype consists of a stack of individually patterned thin films that form a cell chamber conducive to maintaining and recording the electrical activity of a long-term three-dimensional network of rat cortical neurons. Silicon electrode layers contain a polymer grid for neural branching, growth, and network formation. Along the walls of these electrode layers lie exposed gold electrodes which permit recording and stimulation of the neuronal electrical activity. Silicone elastomer microfluidic layers provide a means for loading dissociated neurons into the structure and serve as the artificial vasculature for nutrient supply and aeration. The fluidic layers also serve as insulation for the micro-electrodes. Cells have been shown to survive in the 3D MEA for up to 28 days, with spontaneous and evoked electrical recordings performed in that time. The micro-fluidic capability was demonstrated by flowing in the drug tetrotodoxin to influence the activity of the culture. IntroductionPlanar multi-electrode arrays (MEAs) have been developed for neural applications including brain slice recording [1,2] and dissociated cultures [3,4], to the point where commercial devices are readily available for the study of two-dimensional (2D) or monolayer networks of neurons. There is growing interest in developing three-dimensional (3D) systems that add greater fidelity to these models of the brain. In fact, many cell types have been cultured in both 2D and 3D, and significant differences in behavior have been observed [5][6][7]. In vitro micro-cavity impedance studies have been performed on 3D multi-cellular spheroids [8]. Compared to monolayer cultures, the spheroids better approximate the in vivo cell-cell and cellextracellular matrix contacts that impact cell growth, differentiation, and programmed cell death [9]. The guiding hypotheses of this work are that neuronal culture systems are more realistic in 3D, inferences drawn from 3D cultures are more likely to be valid in vivo, and the development of 3D MEAs would enable advances in this science.The state-of-the-art in 3D arrays for in vivo use includes microwire arrays [10] and silicon multi-electrode probes (e.g., arrays from Michigan [11] and Utah [12]). One group has reported a design for dual-side and double-layer electrode arrays that have been successfully tested in brain tissue samples [13]. It is possible, but sub-optimal, to insert these probes into a 3D in vitro sample. These devices do not include the capability for fluid exchange that is required by in vitro samples and do not have adequate surface area for culturing cells in a liquid media.Pr...

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.

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Characterization of the spontaneous and evoked activity of patterned biological neural networks and discovery of the underlying mechanisms is a major step towards designing functional networks for biosensor, biocomputing and neural prosthetic applications. Here we report on the addition of bicuculline methiodide, a GABA(A) antagonist, to a low density network of embryonic hippocampal neurons linearly patterned on top of planar microelectrode arrays. Our investigation of the differences in the spontaneous and evoked activities before and after drug addition revealed two distinct behavioral trends. One group of electrodes (Group II) showed a marked increase in spontaneous spike rate, burst rate, and burst duration whereas the other (Group I) was unaffected. Likewise, the reliability of evoked activity was observed to be significantly lower in the presence of bicuculline if the stimulating electrode belonged to Group I as compared with Group II. We discuss how these findings might suggest the existence of cells at various stages of maturity in our culture system.