Microdevice for directional axodendritic connectivity between micro 3D neuronal cultures.

Neuronal cultures are widely used in neuroscience research. However, the randomness of circuits in conventional cultures prevents accurate in vitro modeling of cortical development and of the pathogenesis of neurological and psychiatric disorders. A basic feature of cortical circuits that is not captured in standard cultures of dissociated cortical cells is directional connectivity. In this work, a polydimethylsiloxane (PDMS)-based device that achieves directional connectivity between micro 3D cultures is demonstrated. The device consists of through-holes for micro three-dimensional (µ3D) clusters of cortical cells connected by microtrenches for axon and dendrite guidance. The design of the trenches relies in part on the concept of axonal edge guidance, as well as on the novel concept of specific dendrite targeting. This replicates dominant excitatory connectivity in the cortex, enables the guidance of the axon after it forms a synapse in passing (an "en passant" synapse), and ensures that directional selectivity is preserved over the lifetime of the culture. The directionality of connections was verified morphologically and functionally. Connections were dependent on glutamatergic synapses. The design of this device has the potential to serve as a building block for the reconstruction of more complex cortical circuits in vitro.

Micro Three-Dimensional Neuronal Cultures Generate Developing Cortex-Like Activity Patterns.

Studies aimed at neurological drug discovery have been carried out both in vitro and in vivo. In vitro cell culture models have showed potential as drug testing platforms characterized by high throughput, low cost, good reproducibility and ease of handling and observation. However, in vitro neuronal culture models are facing challenges in replicating in vivo-like activity patterns. This work reports an in vitro culture technique that is capable of producing micro three-dimensional (µ3D) cultures of only a few tens of neurons. The µ3D cultures generated by this method were uniform in size and density of neurons. These µ3D cultures had complex spontaneous synchronized neuronal activity patterns which were similar to those observed in the developing cortex and in much larger 3D cultures, but not in 2D cultures. Bursts could be reliably evoked by stimulation of single neurons. Synchronized bursts in µ3D cultures were abolished by inhibitors of glutamate receptors, while inhibitors of GABAA receptors had a more complex effect. This pharmacological profile is similar to bursts in neonatal cortex. Since large numbers of reproducible µ3D cultures can be created and observed in parallel, this model of the developing cortex may find applications in high-throughput drug discovery experiments.

Estimation of the physical properties of neurons and glial cells using dielectrophoresis crossover frequency.

We successfully determine the ranges of dielectric permittivity, cytoplasm conductivity, and specific membrane capacitance of mouse hippocampal neuronal and glial cells using dielectrophoresis (DEP) crossover frequency (CF). This methodology is based on the simulation of CF directly from the governing equation of a dielectric model of mammalian cells, as well as the measurements of DEP CFs of mammalian cells in different suspension media with different conductivities, based on a simple experimental setup. Relationships between the properties of cells and DEP CF, as demonstrated by theoretical analysis, enable the simultaneous estimation of three properties by a straightforward fitting procedure based on experimentally measured CFs. We verify the effectiveness and accuracy of this approach for primary mouse hippocampal neurons and glial cells, whose dielectric properties, previously, have not been accurately determined. The estimated neuronal properties significantly narrow the value ranges available from the literature. Additionally, the estimated glial cell properties are a valuable addition to the scarce information currently available about this type of cell. This methodology is applicable to any type of cultured cell that can be subjected to both positive and negative dielectrophoresis.

Separation and assisted patterning of hippocampal neurons from glial cells using positive dielectrophoresis.

In this work, we describe the separation of embryonic mouse hippocampal neurons from glial cells using a positive dielectrophoresis (DEP) process. Here, we have implemented a cell trapping-favorable, cell suspension solution with low conductivity. It enables positive dielectrophoresis for hippocampal neurons (thereby attracting them to the electrodes), while resulting in negative dielectrophoresis for glial cells (repelling them from the electrodes). We have systematically performed a mathematical simulation and analysis to anticipate the DEP frequency at which hippocampal neurons and glial cells are separated. Simulated DEP crossover frequencies have been experimentally verified, and new, refined glial dielectric and physical properties are suggested that better reflect the experimental results obtained. DEP movements of neurons and glial cells in targeted separation media were experimentally analyzed, under the specified electric signal. Additionally, we have confirmed our modeling results by selectively trapping neurons over electrodes on a custom-made, multi-electrode array (MEA), resulting in active recruitment of neurons over the stimulation and recording sites. This technique is a valuable addition to the toolbox for creating more functional and versatile multi-electrode arrays.

Multi-electrode array capable of supporting precisely patterned hippocampal neuronal networks.

Accurate positioning of primary mouse hippocampal neurons on electrodes enables the recording from and stimulation of specified individual neurons on a multi-electrode array (MEA). In this work, positive dielectrophoresis (DEP) is applied to actively recruit hippocampal neurons to the electrodes of a MEA, whereas microstructures such as chambers and trenches are created to effectively define a patterned neuronal network. We present here the effective pretreatment methods, to improve cytocompatibility of cured thin SU-8 epoxy, commonly used in the fabrication of MEAs. The functionality of our novel MEA is proven by the successful recording of spontaneous and stimulated neuronal potentials from primary hippocampal neurons, including the propagation of evoked neuronal bursts between electrodes.

Cell patterning using molecular vapor deposition of self-assembled monolayers and lift-off technique.

This paper reports a precise, live cell-patterning method by means of patterning a silicon or glass substrate with alternating cytophilic and cytophobic self-assembled monolayers (SAMs) deposited via molecular vapor deposition. Specifically, a stack of hydrophobic heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane SAMs and a silicon oxide adhesion layer were patterned on the substrate surface, and a hydrophilic SAM derived from 3-trimethoxysilyl propyldiethylenetriamine was coated on the remaining non-treated areas on the substrate surface to promote cell growth. The primary characteristics of the reported method include: (i) single-cell resolution; (ii) easy alignment of the patterns with the pre-existing patterns on the substrate; (iii) easy formation of nanoscale patterns (depending on the exposure equipment); (iv) long shelf life of the substrate pattern prior to cell culturing; (v) compatibility with conventional, inverted, optical microscopes for simple visualization of patterns formed on a glass wafer; and (vi) the ability to support patterned cell (osteoblast) networks for at least 2 weeks. Here, we describe the deposition technique and the characterization of the deposited layers, as well as the application of this method in the fabrication of multielectrode arrays supporting patterned neuronal networks.

Precise cell patterning using cytophobic self-assembled monolayer deposited on top of semi-transparent gold.

This paper reports a simple and effective method for cell patterning by using a self-assembled monolayer (SAM)-treated glass surface which is surrounded by semi-transparent gold coated with another type of SAM. Specifically, a hydrophobic SAM, derived from 1-hexadecanethiol (HDT), was coated on the gold surface to prevent cell growth, and a hydrophilic SAM, derived from 3-trimethoxysilyl propyl-diethylenetriamine (DETA), was coated on the exposed glass surface to promote cell growth. The capabilities of this technique are as follows: 1) single-cell resolution, 2) easy alignment of the cell patterns to the structures already existing on the substrate, 3) visualization and verification of the predefined cytophobic/cytophilic pattern prior to cell growth, and 4) convenient monitoring cell growth at the same location for an extended long term period of time. Whereas a number of earlier techniques have demonstrated the single cell resolution, or visualization and verification of the cytophobic/cytophilic patterns prior to cell growth, we believe that our technique is unique in possessing all of these beneficial qualities at the same time. The distinguishing characteristic of our technique is, however, that the use of semi-transparent Cr/Au film allows for convenient brightfield pattern visualization and offers an advantage over previously developed methods which require fluorescent imaging. We have successfully demonstrated the patterning of four different kinds of cells using this technique: immortalized mouse hypothalamic neurons (GT1-7), mouse osteoblast cells (MC3T3), mouse fibroblast cells (NIH3T3) and primary rat hippocampal neurons. This study was performed with a specific ultimate application-the creation of a multi electrode array (MEA) with predefined localization of cell bodies on top of the electrodes, as well as predefined patterns for cell extensions to grow in between the electrodes. With that goal in mind, we have also determined critical parameters for patterning of each of these cell types, such as the minimum size of a cell-adherent island for exclusively anchoring one cell or two cells, as well as the width of the cytophilic pathway between two islands that enables cell extensions to grow, while preventing the anchoring of the cell bodies. Additionally, we have provided statistical analysis of the occupancy for various sizes and shape of cell-anchoring islands. As demonstrated here, we have developed a novel and reliable cell patterning technique, which can be utilized in various applications, such as biosensors or tissue engineering.

Spectral characterization of the voltage-sensitive dye di-4-ANEPPDHQ applied to probing live primary and immortalized neurons.

Spectral properties of a recently developed voltage-sensitive dye, di-4-ANEPPDHQ, were characterized as the dye was dissolved in the solvent dimethyl sulfoxide as the stock solution, in Hank's buffered salt solution as the staining solution, and bound to the plasma membrane of primary rat hippocampal neurons and immortalized mouse hypothalamic neurons (GT1-7) in vitro. Their dependence on the local chemical and electrical environment of dye molecules was determined. The excitation and emission peaks are 479 nm and 570 nm for the stained primary neurons, and 476 nm and 585 nm for the stained immortalized neurons. The excitation and emission bands of the stained GT1-7 neurons, defined as 50% peak intensity, are 429-516 nm and 544-648 nm, respectively.

Effect of three-phase contact line topology on dynamic contact angles on heterogeneous surfaces.

Cassie-Baxter theory has traditionally been used to study liquid drops in contact with microstructured surfaces. The Cassie-Baxter theory arises from a minimization of the global Gibbs free energy of the system but does not account for the topology of the three-phase contact line. We experimentally compare two situations differing only in the microstructure of the roughness, which causes differences in contact line topology. We report that the contact angle is independent of area void fraction for surfaces with microcavities, which correspond to situations when the advancing contact line is continuous. This result is in contrast with Cassie-Baxter theory, which uses area void fraction as the determining parameter, regardless of the type of roughness.