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.

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.

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.

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.

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.