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Immersion photolithography of electrospun SU-8 nanofibers followed by carbonization has been demonstrated for 3-D carbon nanofibrous microelectrodes. The process and resultant structures offer unique advantages: 1) precise patterning of nanofibrous microstructures via refractive index matching immersion photolithography; 2) good cell/neuron adhesion characteristic of the resultant scaffold attributed to nanomorphology in 100’s nanometer scale; 3) high electrical conductivity of the carbonous microelectrodes appropriate for neural stimulation and signal detection; and 4) biocompatibility of the carbon nanofibers. Immiscible refractive index matching liquid, such as oil ( ${n} _{\mathrm {oil}}=1.47$ ), has been applied to the nonhomogeneous nanofiber stack consisting of SU-8 nanofibers ( $n_{\mathrm {SU-8}}=1.67$ ) and air ( $n_{\mathrm {air}}=1$ ) before ultraviolet light exposure, which greatly suppresses optical diffraction and scattering effects, resulting in high aspect ratio 3-D nanofibrous microstructures and enhanced patterning resolution. The aspect ratio of the fabricated 3-D structures is increased from 0.26 to 0.89 and the contrast ratio from 0.56 to 0.96, compared with ones from the nonimmersion process. Ray tracing simulation taking into account diffraction effects in nanofibrous media has been discussed. Microelectrode arrays consisting of integrated nanofibers and thin-film carbon structures are implemented for in vitro neuron culture experiments. Experimental results of structure’s surface roughness, electrical conductivity, and cell viability on them are detailed. [2014-0160]

This paper summarizes the panel discussion at the IEEE Engineering in Medicine and Biology Point-of-Care Healthcare Technology Conference (POCHT 2013) held in Bangalore India from Jan 16–18, 2013. Modern medicine has witnessed interdisciplinary technology innovations in healthcare with a continuous growth in life expectancy across the globe. However, there is also a growing global concern on the affordability of rapidly rising healthcare costs. To provide quality healthcare at reasonable costs, there has to be a convergence of preventive, personalized, and precision medicine with the help of technology innovations across the entire spectrum of point-of-care (POC) to critical care at hospitals. The first IEEE EMBS Special Topic POCHT conference held in Bangalore, India provided an international forum with clinicians, healthcare providers, industry experts, innovators, researchers, and students to define clinical needs and technology solutions toward commercialization and translation to clinical applications across different environments and infrastructures. This paper presents a summary of discussions that took place during the keynote presentations, panel discussions, and breakout sessions on needs, challenges, and technology innovations in POC technologies toward improving global healthcare. Also presented is an overview of challenges and trends in developing and developed economies with respect to priority clinical needs, technology innovations in medical devices, translational engineering, information and communication technologies, infrastructure support, and patient and clinician acceptance of POC healthcare technologies., This paper summarizes the panel discussion at the IEEE Engineering in Medicine and Biology Point of Care Healthcare Technology Conference (POCHT 2013) held in Bangalore India from Jan 16-18, 2013.

Different pairs of adjacent sub-regions of rat hippocampal neurons were co-cultured within two-well devices on micro-electrode arrays (MEAs). Micro-tunnels allowed passage of axons, strong structural connectivity, and information transmission between wells. Paired-pulse stimuli were delivered at each electrode to increase the probability of the excitation without overstimulation. Because of the micro-channel construction, the electrodes inside these tunnels pick up more activity than those within each well. In particular, the strong axonal inputs from the sources of native feed-forward EC-DG and CA3-CA1 co-cultures evoked more target activity than the feed-back networks. In contrast, the evoked target activity in the native feedforward DG-CA3 networks was lower than the reverse flow of information.

To better understand encoding and decoding of stimulus information in two specific hippocampal sub-regions, we isolated and co-cultured rat primary dentate gyrus (DG) and CA3 neurons within a two-chamber device with axonal connectivity via micro-tunnels. We tested the hypothesis that, in these engineered networks, decoding performance of stimulus site information would be more accurate when stimuli and information flow occur in anatomically correct feed-forward DG to CA3 vs. CA3 back to DG. In particular, we characterized the neural code of these sub-regions by measuring sparseness and uniqueness of the responses evoked by specific paired-pulse stimuli. We used the evoked responses in CA3 to decode the stimulation sites in DG (and vice-versa) by means of learning algorithms for classification (support vector machine, SVM). The device was placed over an 8 × 8 grid of extracellular electrodes (micro-electrode array, MEA) in order to provide a platform for monitoring development, self-organization, and improved access to stimulation and recording at multiple sites. The micro-tunnels were designed with dimensions 3 × 10 × 400 μm allowing axonal growth but not migration of cell bodies and long enough to exclude traversal by dendrites. Paired-pulse stimulation (inter-pulse interval 50 ms) was applied at 22 different sites and repeated 25 times in each chamber for each sub-region to evoke time-locked activity. DG-DG and CA3-CA3 networks were used as controls. Stimulation in DG drove signals through the axons in the tunnels to activate a relatively small set of specific electrodes in CA3 (sparse code). CA3-CA3 and DG-DG controls were less sparse in coding than CA3 in DG-CA3 networks. Using all target electrodes with the three highest spike rates (14%), the evoked responses in CA3 specified each stimulation site in DG with optimum uniqueness of 64%. Finally, by SVM learning, these evoked responses in CA3 correctly decoded the stimulation sites in DG for 43% of the trials, significantly higher than the reverse, i.e., how well-recording in DG could predict the stimulation site in CA3. In conclusion, our co-cultured model for the in vivo DG-CA3 hippocampal network showed sparse and specific responses in CA3, selectively evoked by each stimulation site in DG.

I recently had the opportunity to give a short talk describing the IEEE Engineering in Medicine and Biology Society (EMBS) to students and faculty of the University of Florida. While looking over background material, I came to appreciate how deep and long the roots of our Society travel and how strong and wide it currently stands.

Anatomical and behavioral studies, and in vivo and slice electrophysiology of the hippocampus suggest specific functions of the dentate gyrus (DG) and the CA3 subregions, but the underlying activity dynamics and repeatability of information processing remains poorly understood. To approach this problem, we engineered separate living networks of the DG and CA3 neurons that develop connections through 51 tunnels for axonal communication. Growing these networks on top of an electrode array enabled us to determine whether the subregion dynamics were separable and repeatable. We found spontaneous development of polarized propagation of 80% of the activity in the native direction from DG to CA3 and different spike and burst dynamics for these subregions. Spatial-temporal differences emerged when the relationships of target CA3 activity were categorized with to the number and timing of inputs from the apposing network. Compared to times of CA3 activity when there was no recorded tunnel input, DG input led to CA3 activity bursts that were 7× more frequent, increased in amplitude and extended in temporal envelope. Logistic regression indicated that a high number of tunnel inputs predict CA3 activity with 90% sensitivity and 70% specificity. Compared to no tunnel input, patterns of >80% tunnel inputs from DG specified different patterns of first-to-fire neurons in the CA3 target well. Clustering dendrograms revealed repeating motifs of three or more patterns at up to 17 sites in CA3 that were importantly associated with specific spatial-temporal patterns of tunnel activity. The number of these motifs recorded in 3 min was significantly higher than shuffled spike activity and not seen above chance in control networks in which CA3 was apposed to CA3 or DG to DG. Together, these results demonstrate spontaneous input-dependent repeatable coding of distributed activity in CA3 networks driven by engineered inputs from DG networks. These functional configurations at measured times of activation (motifs) emerge from anatomically accurate feed-forward connections from DG through tunnels to CA3.

Transient propagation of information across neuronal assembles is thought to underlie many cognitive processes. However, the nature of the neural code that is embedded within these transmissions remains uncertain. Much of our understanding of how information is transmitted among these assemblies has been derived from computational models. While these models have been instrumental in understanding these processes they often make simplifying assumptions about the biophysical properties of neurons that may influence the nature and properties expressed. To address this issue we created an in vitro analog of a feed-forward network composed of two small populations (also referred to as assemblies or layers) of living dissociated rat cortical neurons. The populations were separated by, and communicated through, a microelectromechanical systems (MEMS) device containing a strip of microscale tunnels. Delayed culturing of one population in the first layer followed by the second a few days later induced the unidirectional growth of axons through the microtunnels resulting in a primarily feed-forward communication between these two small neural populations. In this study we systematically manipulated the number of tunnels that connected each layer and hence, the number of axons providing communication between those populations. We then assess the effect of reducing the number of tunnels has upon the properties of between-layer communication capacity and fidelity of neural transmission among spike trains transmitted across and within layers. We show evidence based on Victor-Purpura's and van Rossum's spike train similarity metrics supporting the presence of both rate and temporal information embedded within these transmissions whose fidelity increased during communication both between and within layers when the number of tunnels are increased. We also provide evidence reinforcing the role of synchronized activity upon transmission fidelity during the spontaneous synchronized network burst events that propagated between layers and highlight the potential applications of these MEMs devices as a tool for further investigation of structure and functional dynamics among neural populations.

In this study we created four network topologies composed of living cortical neurons and compared resultant structural-functional dynamics including the nature and quality of information transmission. Each living network was composed of living cortical neurons that were created using microstamping of adhesion promoting molecules and each was “designed” with different levels of convergence embedded within each structure. Networks were cultured over a grid of electrodes that permitted detailed measurements of neural activity at each node in the network. Of the topologies we tested, the “Random” networks in which neurons connect based on their own intrinsic properties transmitted information embedded within their spike trains with higher fidelity relative to any other topology we tested. Within our patterned topologies in which we explicitly manipulated structure, the effect of convergence on fidelity was dependent on both topology and time-scale (rate versus temporal coding). At long time scales associated with rate based coding the simple linear 2D or “line” networks produced the highest fidelity during transmission among neurons (nodes) followed by 4D (4 connections per node) and 8D (8 connections per node) grid networks. By contrast, at more precise temporal scales it was the increased convergence within the 8D topology that now resulted in the highest fidelity followed by 4D and 2D networks. A more detailed examination using tools from network analysis revealed that these changes in fidelity were also associated with a number of other structural properties including a node’s degree, degree-degree correlations, path length, and clustering coefficients. Whereas information transmission was apparent among nodes with few connections, the greatest transmission fidelity was achieved among the few nodes possessing the highest number of connections (high degree nodes or putative hubs). These results provide a unique view into the relationship between structure and its affect on transmission fidelity, at least within these small neural populations with defined network topology. They also highlight the potential role of tools such as microstamp printing and microelectrode array recordings to construct and record from arbitrary network topologies to provide a new direction in which to advance the study of structure-function relationships.

Toward the goal of reproducible live neuronal networks, we investigated the influence of substrate patterns on neuron compliance and network activity. We optimized process parameters of micro-contact printing for reproducible geometric patterns of 10 µm wide lines of polylysine with 4, 6, or 8 connections at a constant square array of nodes overlying the recording electrodes of a multielectrode array (MEA). We hypothesized that an increase in node connections would give the network more inputs resulting in higher neuronal outputs as network spike rates. We also chronically stimulated these networks during development and added astroglia to enhance network activity. Our results show that despite frequent localization of neuron somata over the electrodes, the number of spontaneously active electrodes was reduced 3-fold compared to random networks, independent of pattern complexity. Of the electrodes active, the overall spike rate was independent of pattern complexity, consistent with homeostasis of activity. Lower mean burst rates were seen with higher levels of pattern complexity; however, burst durations increased 1.6-fold with pattern complexity (n=6027 bursts, p

Background: Feline oral squamous cell carcinoma (SCC) carries a very poor prognosis with traditional treatments. Hypothesis/Objectives: To examine the effectiveness of adding carboplatin to a previously published accelerated radiation protocol in the treatments of oral SCC in cats. Animals: Thirty-one cases of oral SCC in cats. Tumor sites included lingual (n = 9), mandible (n = 10), maxilla (n = 7), tonsil (n = 4), and cheek (n = 1). Methods: Prospective trial using a planned radiation protocol consisting of 14 fractions of 3.5 Gy given within a 9-day period with the addition of carboplatin given at 90–100 mg/m2 on day 1 and day 4.5. Treatments were twice daily with a 6-hour delay between treatments. All cats presenting with oral SCC without evidence of distant metastasis were eligible. Results: Median survival for all cats was 163 days (range 53–770 days) with a mean of 319 ± 53 days with significant predictors of survival being site (P= .004) and whether there was a complete response at 30 days (P= .001). Cats with tumors of tonsil origin or cheek responded best to therapy and were long-term survivors with a mean survival of 724 days and the median had not been reached because of continued survival of 4 cats. Conclusions and Clinical Importance: This protocol offers an aggressive yet tolerable treatment of oral SCC in cats that might offer improved survival as compared with previously reported treatments. The long-term survival of cats with tonsillar SCC has not been reported previously.

In vitro microelectrode array (MEA) technology has evolved into a widely used and effective methodology to study cultured neural networks. An MEA forms a unique electrical interface with the cultured neurons in that neurons are directly grown on top of the electrode (neuron-on-electrode configuration). Theoretical models and experimental results suggest that physical configuration and biological environments of the cell-electrode interface play a key role in the outcome of neural recordings, such as yield of recordings, signal shape, and signal-to-noise ratio. Recent interdisciplinary approaches have shown that MEA performance can be enhanced through novel nanomaterials, structures, surface chemistry, and biotechnology. In vitro and in vivo neural interfaces share some common factors, and in vitro neural interface issues can be extended to solve in vivo neural interface problems of brain-machine interface or neuromodulation techniques.

Objective Functions ascribed to the hippocampal sub-regions for encoding episodic memories include the separation of activity patterns propagated from the entorhinal cortex (EC) into the dentate gyrus (DG) and pattern completion in CA3 region. Since a direct assessment of these functions is lacking at the level of specific axonal inputs, our goal is to directly measure the separation and completion of distinct axonal inputs in engineered pairs of hippocampal sub-regional circuits. Approach We co-cultured EC-DG, DG-CA3, CA3-CA1 or CA1-EC neurons in a two-chamber PDMS device over a micro-electrode array (MEA60), inter-connected via distinct axons that grow through the micro-tunnels between the compartments. Taking advantage of the axonal accessibility, we quantified pattern separation and completion of the evoked activity transmitted through the tunnels from source into target well. Since pattern separation can be inferred when inputs are more correlated than outputs, we first compared the correlations among axonal inputs with those of target somata outputs. We then compared, in an analog approach, the distributions of correlation distances between rate patterns of the axonal inputs inside the tunnels with those of the somata outputs evoked in the target well. Finally, in a digital approach, we measured the spatial population distances between binary patterns of the same axonal inputs and somata outputs. Main results We found the strongest separation of the propagated axonal inputs when EC was axonally connected to DG, with a decline in separation to CA3 and to CA1 for both rate and digital approaches. Furthermore, the digital approach showed stronger pattern completion in CA3, then CA1 and EC. Significance To the best of our knowledge, these are the first direct measures of pattern separation and completion for axonal transmission to the somata target outputs at the rate and digital population levels in each of four stages of the EC-DG-CA3-CA1 circuit.

Technology has advanced to where it is possible to design and grow - with predefined geometry and surprisingly good fidelity - living networks of neurons in culture dishes. Here we overview the elements of design, emphasizing the lithographic techniques that alter the cell culture surface which in turn influences the attachment and growth of the neural networks. Advanced capability in this area makes it possible to design networks of desired complexity. Other issues addressed include the influence of glial cells and media on activity and the potential for extending the designs into three dimensions. Investigators are advancing the art and science of analyzing and controlling through stimulation the function of the neural networks, including the ability to take advantage of their geometric form in order to influence functional properties.

This study investigated a novel multi-electrode-array (MEA) design capable of long-term and highly selective recordings of axonal signals using PDMS microtunnels. We successfully grew neurons in culture so that only axons extended through narrow (10 microm wide by 3 microm high) and long (750 microm) microtunnels under which multiple electrodes were integrated. This permitted the recording of relatively large (up to 200 microV) electrical signals, including the propagation speed and direction of these travelling action potentials. To further demonstrate the operation of the device as a diagnostic tool for drug screening assays, the drug mepivacaine was applied in washout experiments. Here, we identified significant changes in mean spiking rate and conduction velocity.

To fully exploit the recording capabilities provided by current and future generations of multi-electrode arrays, some means to eliminate the residual charge and subsequent artifacts generated by stimulation protocols is required. Custom electronics can be used to achieve such goals, and by making them scalable, a large number of electrodes can be accessed in an experiment. In this work, we present a system built around a custom 16-channel IC that can stimulate and record, within 3 ms of the stimulus, on the stimulating channel, and within 500 mus on adjacent channels. This effectiveness is achieved by directly discharging the electrode through a novel feedback scheme, and by shaping such feedback to optimize electrode behavior. We characterize the different features of the system that makes such performance possible and present biological data that show the system in operation. To enable this characterization, we present a framework for measuring, classifying, and understanding the multiple sources of stimulus artifacts. This framework facilitates comparisons between artifact elimination methodologies and enables future artifact studies.

Microelectrode arrays (MEAs) are commonly utilized for stimulating and recording extracellular electrical signals including both local field and action potentials in both in-vitro and in-vivo neural studies. This work demonstrates the feasibility of 3D microelectrode arrays using a novel carbon nanomaterial, electrospun carbon nanofiber (CNF). CNF MEAs impedance is characterized and compared to that of carbon nanotube MEAs and commercial TiN MEAs. An in-vitro culture of CNF electrodes are performed using E18 cortical neurons and analyzed for cell interaction. With these electrodes we are able to detect extracellular neural signals including action potentials from single neurons from an array of CNF electrodes embedded within the substrate of an MEA.

Carbon nanomaterials have become increasingly popular microelectrode materials for neuroscience applications. Here we study how the scale of carbon nanotubes and carbon nanofibers affect neural viability, outgrowth, and adhesion. Carbon nanotubes were deposited on glass coverslips via a layer-by-layer method with polyethylenimine (PEI). Carbonized nanofibers were fabricated by electrospinning SU-8 and pyrolyzing the nanofiber depositions. Additional substrates tested were carbonized and SU-8 thin films and SU-8 nanofibers. Surfaces were O2-plasma treated, coated with varying concentrations of PEI, seeded with E18 rat cortical cells, and examined at 3, 4, and 7 days in vitro (DIV). Neural adhesion was examined at 4 DIV utilizing a parallel plate flow chamber. At 3 DIV, neural viability was lower on the nanofiber and thin film depositions treated with higher PEI concentrations which corresponded with significantly higher zeta potentials (surface charge); this significance was drastically higher on the nanofibers suggesting that the nanostructure may collect more PEI molecules, causing increased toxicity. At 7 DIV, significantly higher neurite outgrowth was observed on SU-8 nanofiber substrates with nanofibers a significant fraction of a neuron's size. No differences were detected for carbonized nanofibers or carbon nanotubes. Both carbonized and SU-8 nanofibers had significantly higher cellular adhesion post-flow in comparison to controls whereas the carbon nanotubes were statistically similar to control substrates. These data suggest a neural cell preference for larger-scale nanomaterials with specific surface treatments. These characteristics could be taken advantage of in the future design and fabrication of neural microelectrodes.

Surface chemistry is one of the main factors that contributes to the longevity and compliance of cell patterning. Two to three weeks are required for dissociated, embryonic rat neuronal cultures to mature to the point that they regularly produce spontaneous and evoked responses. Though proper surface chemistry can be achieved through the use of covalent protein attachment, often it is not maintainable for the time periods necessary to study neuronal growth. Here we report a new and effective covalent linking approach using (3-glycidoxypropyl) trimethoxysilane (3-GPS) for creating long term neuronal patterns. Micrometer scale patterns of cell adhesive proteins were formed using microstamping; hippocampal neurons, cultured up to 1 month, followed those patterns. Cells did not grow on unmodified 3-GPS surfaces, producing non-permissive regions for the long-term cell patterning. Patterned neuronal networks were formed on two different types of MEA (polyimide or silicon nitride insulation) and maintained for 3 weeks. Even though the 3-GPS layer increased the impedance of metal electrodes by a factor of 2-3, final impedance levels were low enough that low noise extracellular recordings were achievable. Spontaneous neural activity was recorded as early as 10 days in vitro. Neural recording and stimulation were readily achieved from these networks. Our results showed that 3-GPS could be used on surfaces to immobilize biomolecules for a variety of neural engineering applications.

We report the use of a gold coating on microelectrode arrays (MEAs) to enable the use of the relatively reliable surface modification chemistry afforded by alkanethiol self-assembled monolayers (SAMs). The concept is simple and begins with planar MEAs, which are commercially available for neuronal cell culture and for brain slice studies. A gold film, with an intermediate adhesive layer of titanium, is deposited over the insulation of an existing MEA in a manner so as to be thin enough for transmission light microscopy as well as to avoid electrical contact to the electrodes. The alkanethiol-based linking chemistry is then applied for the desired experimental purpose. Here we show that polylysine linked to alkanethiol SAM can control the geometry of an in vitro hippocampal neuronal network grown on the MEA. Furthermore, recordings of neuronal action potentials from random and patterned networks suggest that the gold coating does not significantly alter the electrode properties. This design scheme may be useful for increasing the number of neurons located in close proximity to the electrodes. Realization of in vitro neuronal circuits on MEAs may significantly benefit basic neuroscience studies, as well as provide the insight relevant to applications such as neural prostheses or cell-based biosensors. The gold coating technique makes it possible to use the rich set of thiol-based surface modification techniques in combination with MEA recording.

We report the design and application of a Micro Electro Mechanical Systems (MEMs) device that permits investigators to create arbitrary network topologies. With this device investigators can manipulate the degree of functional connectivity among distinct neural populations by systematically altering their geometric connectivity in vitro. Each polydimethylsilxane (PDMS) device was cast from molds and consisted of two wells each containing a small neural population of dissociated rat cortical neurons. Wells were separated by a series of parallel micrometer scale tunnels that permitted passage of axonal processes but not somata; with the device placed over an 8 × 8 microelectrode array, action potentials from somata in wells and axons in microtunnels can be recorded and stimulated. In our earlier report we showed that a one week delay in plating of neurons from one well to the other led to a filling and blocking of the microtunnels by axons from the older well resulting in strong directionality (older to younger) of both axon action potentials in tunnels and longer duration and more slowly propagating bursts of action potentials between wells. Here we show that changing the number of tunnels, and hence the number of axons, connecting the two wells leads to changes in connectivity and propagation of bursting activity. More specifically, the greater the number of tunnels the stronger the connectivity, the greater the probability of bursting propagating between wells, and shorter peak-to-peak delays between bursts and time to first spike measured in the opposing well. We estimate that a minimum of 100 axons are needed to reliably initiate a burst in the opposing well. This device provides a tool for researchers interested in understanding network dynamics who will profit from having the ability to design both the degree and directionality connectivity among multiple small neural populations.

During synaptogenesis information exchanged locally between synaptic partners results in precise alignment of morphological and molecular specializations. For example, agrin derived from motoneurons induces localized postsynaptic differentiation at the neuromuscular synapse. Similar information molecules are thought to act at other synapses; however, techniques for directly evaluating synaptogenic activities of such molecules are lacking. Here we use agrin-induced differentiation as a model system to validate a novel approach for characterizing synaptogenic molecules. Proteins are patterned with micron scale resolution on glass coverslips by covalent microcontact printing and these substrates are used for cell culture. Postsynaptic molecules accumulate specifically at sites of contact between muscle cells and patterned agrin: a response which is quantifiable. Our results demonstrate that microcontact printing is applicable to the analysis of cellular response to locally immobilized information molecules.

Crucial to long-term stability of neuronal micropatterns is functional retention of the underlying substratum while exposed to cell culture conditions. We report on the ability of covalently bound PEG films in long-term cell culture to continually retard protein adhesion and cell growth. PDMS microstamps were used to create poly-d-lysine (PDL) substrates permissive to cell attachment and growth, and polyethylene glycol (PEG) substrates were used to minimize protein and cell adhesion. Film thickness was measured using null ellipsometry and atomic force microscopy (AFM). Organosilane film structure was examined using Fourier transform infrared (FT-IR) spectroscopy. Long-term film stability in cell culture conditions was tested by immersion in 0.1 m sodium phosphate buffer pH 7.4 for up to one month. Null ellipsometry and water contact measurements indicated that organosilane films were stable up to one month, whereas the PEG film thickness declined rapidly after day 25. Hippocampal cells plated at 200 cells/mm2 on uniform PEG substrates gave a steady increase in biofilm thickness on PEG films throughout the culture, possibly from proteins of neuronal origin. We found that all the layers in the cross-linking procedure were stable in cell culture conditions, with the exception of PEG, which degraded after day 25.

For neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either polyethylene glycol (PEG) or the amino acid serine (C/sub 3/H/sub 7/NO/sub 3/) were evaluated. The foreground regions were microstamped with 3-, 5-, or 10-/spl mu/m lines of poly-D-lysine (PDL), which promotes neural attachment and growth. After 29 days in culture the foreground compliance, or the fraction of all live somata which rested on the desired PDL surface, averaged 86% for serine and 90% for PEG, with only a small decline. The background compliance, or the fraction of square areas in the pattern background which were free of neurite extension, declined from highs of 40% and 55% (midculture) to 5.5% and 12% (29 days) for serine and PEG, respectively. Images of the cultures suggest that PEG is significantly more effective as a nonpermissive substrate. The authors conclude that these materials, especially PEG, are adequate for the maintenance of long-term patterned cultures of neurons. They believe that this is the first report of high-quality long-term patterning of cultured neurons.

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.

We have developed a technique for producing patterned biomolecule gradients on glass substrates using microstamping. The technique uses a microfabricated polydimethylsiloxane (PDMS) stamp with micrometer length features to transfer biomolecules to silane-derivatized substrates. The soaking solution for the stamp is subjected to an electric field to electrophoretically produce a gradient in solution and hence on the stamp. To test the efficacy of the procedure we tested substrates stamped with poly-D-lysine by fluorescently labeling the amine groups and measuring the fluorescence. It is possible to modify the steepness of the gradient using both voltage and electrode spacing, and the profile can be modeled using an isoelectric focusing model. Since gradients have been implicated in roles in guidance and patterning of neurons and other cell types, a flexible method to produce biomolecule gradients using microstamping may further extend the usefulness of microstamping to studies of cell patterning and guidance.

Microcontact printing, facilitated by silane linker chemistry and high-relief stamps, creates precise patterns of proteins, which in turn control growth of hippocampal neurons in culture. This additive, multi-mask technique permits several different molecules to be patterned on the same substrate. The covalent linker technology permits relatively long-term (two-week) compliance of neurons to the stamped pattern against a polyethylene glycol background. When polylysine was stamped adjacent to a laminin/polylysine mixture, neural somata and dendrites preferred the polylysine while axons prefer the mixture or the border between the two.