Download PDF Article Open access Published: 04 September 2018 Single-neuronal cell culture and monitoring platform using a fully transparent microfluidic DEP device Hyungsoo Kim1, In-Kyu

Lee1, Kendra Taylor2, Karl Richters3, Dong-Hyun Baek4, Jae Ha Ryu1, Sang June Cho  ORCID: orcid.org/0000-0002-7784-24981, Yei Hwan Jung1, Dong-Wook Park1,5, Joseph Novello4, Jihye Bong1,

Aaron J. Suminski4, Aaron M. Dingle6, Robert H. Blick1, Justin C. Williams  ORCID: orcid.org/0000-0002-8710-74974, Erik W. Dent3 & …Zhenqiang Ma1 Show authors Scientific Reports volume 8,

Article number: 13194 (2018) Cite this article

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Dielectrophoresis using multi-electrode arrays allows a non-invasive interface with biological cells for long-term monitoring of electrophysiological parameters as well as a label-free and

non-destructive technique for neuronal cell manipulation. However, experiments for neuronal cell manipulation utilizing dielectrophoresis have been constrained because dielectrophoresis

devices generally function outside of the controlled environment (i.e. incubator) during the cell manipulation process, which is problematic because neurons are highly susceptible to the

properties of the physiochemical environment. Furthermore, the conventional multi-electrode arrays designed to generate dielectrophoretic force are often fabricated with non-transparent

materials that confound live-cell imaging. Here we present an advanced single-neuronal cell culture and monitoring platform using a fully transparent microfluidic dielectrophoresis device

for the unabated monitoring of neuronal cell development and function. The device is mounted inside a sealed incubation chamber to ensure improved homeostatic conditions and reduced

contamination risk. Consequently, we successfully trap and culture single neurons on a desired location and monitor their growth process over a week. The proposed single-neuronal cell

culture and monitoring platform not only has significant potential to realize an in vitro ordered neuronal network, but also offers a useful tool for a wide range of neurological research

and electrophysiological studies of neuronal networks.

Portrait of intense communications within microfluidic neural networks Article Open access 29 July 2023 NeuroExaminer: an all-glass microfluidic device for whole-brain in vivo imaging in

zebrafish Article Open access 16 June 2020 Introduction

Single-cell analysis has attracted an increasing amount of attention over the past decades, and paves the way for elucidating fundamental biological phenomena such as cellular processes and

heterogeneities1,2. Of particular importance to the field of neuroscience, meticulous studies of single neurons and between spatially isolated neurons provide a better understanding of the

dynamics of functional neuronal networks as well as their fundamental molecular and cellular mechanisms3. This research is essential to push forward personalized treatments of neurological

disorders including epilepsy, Parkinson’s disease, Alzheimer’s disease, and other cognitive and motor disorders4.

To date, various cell manipulation techniques such magnetophoresis, optical tweezers, acoustic means, and dielectrophoresis (DEP), have been explored for the field of single-cell

analysis2,5,6,7,8,9. Among these techniques, DEP, an electrokinetic phenomenon acting on polarizable particles in a non-uniform electric field, benefits from the fact that cells can be

trapped, aligned and patterned without requiring additional elements (i.e. optical device, magnet and light source)5,10,11,12,13,14,15,16. Moreover, DEP provides a healthy environment for

neurons to reside by incorporating electrode structures that are designed to minimize the electric field intensity9.

Nevertheless, the availability of DEP for realizing an in vitro cultured neuronal network is limited by the difficulty of the neuron cultures, which are highly susceptible to the properties

of the physiochemical environment (i.e. pH, osmotic pressure, humidity, and temperature) and infection17,18,19,20. In addition, imaging the morphology and activity of cultured neurons using

inverted microscope is often confounded by the use of non-transparent electrodes and substrate17,18,19,21.

Here, we propose an advanced single-neuronal cell culture and monitoring platform using a fully transparent microfluidic DEP device. This device consists of multi-electrode arrays (MEAs)

made of indium-tin-oxide (ITO) and a polydimethylsiloxane(PDMS) microfluidic chip. To reduce the risk of culture contamination, the device was mounted inside an incubated microscope system.

A target neuron was trapped and released sequentially by an array of ring-shaped electrodes arranged in a row to demonstrate the capabilities of the proposed system. Consequently, we were

able to successfully culture and monitor single-neuronal cells over time. This advanced platform for trapping of single-neuronal cells and monitoring of its electrophysiological parameters

enables novel and detailed neurological studies.

DEP is a translational motion of polarizable particles suspended in a medium induced by a non-uniform electric field12,22. The time-averaged DEP force on a particle in a non-uniform electric

field can be expressed as

where R is the radius of the particle, εm is the relative permittivity of the surrounding medium, Re[fCM(ω)] is the real part of the Clausius-Mossotti (CM) factor, ▽ is the del vector

operator, and Erms is the root-mean-square value of the applied electric field12. For the case of a spherical, homogeneous particle of permittivity εp, the CM factor which describes the

effective polarizability of the particle which varies with the applied frequency23 is given by

}}{{\varepsilon }_{p}^{\ast }+2{\varepsilon }_{m}^{\ast }}$$ (2)

where \({\varepsilon }_{p}^{\ast }\) and \({\varepsilon }_{m}^{\ast }\) are the complex permittivities of the particle and the medium, respectively, with \({\varepsilon }_{p}^{\ast

}={\varepsilon }_{p}-j\frac{{\sigma }_{p}}{\omega }\), and \({\varepsilon }_{m}^{\ast }={\varepsilon }_{m}-j\frac{{\sigma }_{m}}{\omega }\) where εp and εm are the permittivities of the

particle and the medium, respectively, σp and σm are the conductivities of the particle and the medium, respectively, and ω is the angular frequency of the applied electric field. The real

part of the CM factor (Re[fCM(ω)]) has a value between −0.5 and 1 and determines the direction of the DEP force. When the particles that are more polarizable than the surrounding media, the

Re[fCM(ω)] is positive and the particles are attracted to the regions of electric field intensity maxima, positive dielectrophoresis (pDEP), whereas when the particles that are less

polarizable than the media, Re[fCM(ω)] is negative and the particles move toward electric field intensity minima (i.e. repelled from field maxima), negative dielectrophoresis (nDEP). For

neuroscience applications, the nDEP is better suited as it makes possible the use of commonly used neuronal cell culture media due to the conductivity and permittivity of the media being

higher than those of neurons10,11,19 as well as allows neurons to reside in a healthy environment by attracting the neurons to the region of electric field intensity minima.

DiscussionsDevice modeling and simulation

To verify the feasibility of the proposed DEP device for single neuronal cell manipulation, the strength of the electrical field and the direction of the DEP force over the device including

electrodes were numerically solved using finite element simulation software (Comsol Multiphysics 4.2, Comsol Ltd). The amplitude and frequency of the applied voltage in this simulation were

8 Vpp and 10 MHz, respectively. For the designed ring-shaped electrodes, we presumed the electric field distribution to be cylindrically symmetric in any plane orthogonal to the plane of the

array of electrodes.

For the simulation, various physical parameters of the structure and a dipolar model of the DEP force were established. The parameters of the neuronal cell and medium were as follows: the

radius of the cell: 5 µm, permittivity of the cell: 80, cytoplasm permittivity: 7.1 × 10−10 F/m, cytoplasm conductivity: 0.75 S/m, membrane permittivity: 1.8 × 10−12 F/m, membrane

conductivity: 1 × 10−7 S/m and medium permittivity: 7.1 × 10−10 F/m14,19,24,25,26. As for the boundary conditions, applied AC electric potentials were on the ring-shaped electrodes and the

outer surfaces were set to electrical insulation.

Figure 1a shows the distribution of the electric field magnitude (E2) for each trap electrode with an applied signal of 8 Vpp at 10 MHz. As can be seen in this figure, the magnitude of the

electric field changed over the position (x-axis), with a minimum in the center of the ring-shaped trap electrode and maximum at the edge of electrode in the gap between the trap electrode

and surrounding counter electrode. Dielectrophoretic forces (white arrows) are directed toward the center of the ring-shaped electrode and repellent forces are displayed near the surrounding

electrodes. This simulation result indicates that invisible trap formed at the center of the electrode with low electric field magnitude.

Schematic illustrations of trap electrode arrays and its cross-sectional view with numerical simulation results. The color bar shows the electric field intensity (in V/m) for an applied AC

signal. (a) Distribution of the electric field magnitude (in V/m), based on an applied potential of 8 Vpp at 10 MHz, is shown for each trap electrode inside the fluidic channel with

color-scale plot. The white arrows, normalized vectors, indicate the direction of the dielectrophoretic force. The intensity of the applied electric field is maximal in close proximity to

the edge of each ring-shaped electrode and is reduced to its minimum value at the center of the trap zone. (b) Motion trajectories of neurons with a radius of 5 µm under the distribution of

applied electric field magnitude (in V/m). Numerals I-IV correspond to time: (I) Initial distribution of neurons in the domain, (II) position of the neurons after 0.3 s, (III) 1 s, and (IV)

2 s.

The numerical simulation results of the neuron motion tracking at each instant are illustrated in Fig. 1b. Neurons were modeled as blue particles with a radius of 5 µm and distributed

uniformly at the initial stage (I) of the simulation. The blue particles placed near the ring trap were driven toward regions of low field strength and collected in the center of the ring

trap. However, the particles placed outside of the ring trap moved upwards, repelled by the repulsive force over time. This simulation indicates that our microfluidic DEP device is suitable

for single cell manipulation.

To demonstrate the performance of our proposed fully-transparent microfluidic DEP device, single-neuronal cell manipulation was conducted as shown in Fig. 2. The cell trapping process was

carried out inside an incubator and monitored with a built-in CCD camera (DS-Qi1, Nikon, Inc.). The single-neuronal cell trapping process is illustrated in Fig. 2a. The neuronal cells were

trapped and released sequentially by an array of ring electrodes arranged in a row. Details of the neuronal cell positioning process are as follows: (I) A target neuron (red arrowhead) flows

to the 1st electrode. (II) When the target neuron comes in proximity to the 1st electrode, the 1st electrode is energized and the neuron is immobilized in the center of the 1st trap site.

(III) Non-cellular particles (blue arrowhead) are repelled by the 1st electrode and keep flowing while the trapped neuron remains at the 1st trap site (green arrows). (IV) The trapped neuron

is released by turning the 1st electrode off, travels with the flow of the media, and then is trapped again in the center of the 2nd trap site. (V-VI) The neuron is then subsequently

released and trapped in turn by the 3rd and 4th trap sites.

Recorded images of single-neuronal cell manipulation on the array of ring-shaped traps. (a) Incoming neuron (I) entering the 1st trap. (II) The neuron is then immobilized in the 1st trap

electrode against a fluid flow. (III) While the neuron is trapped, a repelled particle continues to move in the flow of media. (IV) The released neuron is captured again in the 2nd trap. (V

and VI) The neuron is trapped in the 3rd and the 4th ring trap in turn. (b) Bouncing motion of the neuron subject to a repulsive force. While the target neuron was trapped in the desired

electrode, an incoming neuron was repelled by DEP force. When the incoming neuron reached the outside of the electrode, the repulsive force pushed the neuron out of the ring. Video is

included in Supplementary Materials (Movie S1).

We also observed neurons being repelled from the electrode. The images in Fig. 2b show the bouncing motion of the neuron which is subject to a repulsive force induced by the nDEP. (I and II)

While the target neuron was immobilized in the ring trap (red arrowhead), another neuron comes in close proximity to the ring trap and it appears to have bounced off the invisible wall

created by the repulsive force (black arrow). (III) The repelled neuron travelled along the invisible wall carried by the flowing media, while the target neuron stays in the trap. The

trapping and bouncing motion of the neuron was in concordance with the particle trajectory simulation results (Fig. 1b). Video of single-neuronal cell manipulation recorded through the

transparent electrode sites can be seen in the Supplementary Materials (Movie S1).

After the cell trapping process, the media was aspirated and replaced with fresh culture media to remove any redundant neurons and cellular debris remaining in the microfluidic chamber and

deliver nutrients to the trapped neurons. The fluid flow was shut down and the system was stabilized for 5 min. Then, the nDEP forces were turned off so that the trapped neurons levitating

above the electrode plane were released from its levitated position and plated down for culture. During the cell culture period, we changed the culture media once a day to supply nutrients

to the neurons by aspirating away approximately half of the media and replacing the amount removed with fresh media. The cultivation images were recorded by a CCD camera integrated inside

the incubator. Red LED illumination was used for phase contrast imaging.

To confirm whether the trapped single neurons settle and grow well on the trap electrode, we monitored the morphological changes of growing neurons for 20 hrs. Figure 3a shows the time-lapse

phase contrast images of neurite outgrowth in an in vitro culture of a trapped single neuron. As can be seen in this figure, the morphology of neuron changed slightly after 1 hr and minor

neurites began to form after 2 hrs. After 4 hrs, we observed that several minor neurites protruded the cell body and continued to extend over time. These results demonstrate the viability of

the technique as we confirmed that a single neuron successfully adhered to the trap electrode and grew well over time.

Images of cultured neurons on trap electrodes. (a) In vitro time-lapse imaging of outgrowth of a single neuron on the trap electrode for 20 h. The trapped neuron was attached on the surface

at the initial stage of the imaging. Time-lapse phase contrast images of a living cortical neuron show outgrowth of neurites. (b) Microscope image of a cultured neuron on a trap electrode

(left). Neuron was fixed at 5 days in vitro (5DIV). Image of neuron immunolabeled for microtubules (red) and actin (green) (right).

All of the neurons cultured on the fabricated devices show the same developmental trajectory. First, neurons exhibited a stereotypical series of events in which they first attach to a

substrate and extend both lamellipodia and filopodia (stage 1). Over time, filopodia merge and form several distinct neurites, all of which contain a growth cone at their tip (stage 2).

Within 48 hours of plating, one of the neurites elongates rapidly to form an axon (stage 3), while the remaining neurites develop slowly into dendrites (stage 4)27. A DIC and a fluorescent

image of a single neuron cultured on the trap electrode are shown in Fig. 3b. Neurons cultured on poly-D-lysine(PDL)-coated DEP devices were fixed at 5 days in vitro (5DIV) and labelled for

microtubules (red) and actin (green) (Fig. 3b). Phalloidin staining at the tips of both neurites and growth cones show typical stage 2 to 3 development, as well as prominent tyrosinated

tubulin staining, which labels dynamic microtubules, in both dendrites and axons28.

We have presented an advanced single-neuronal cell culture and monitoring platform which enables single-neurons to be positioned at a desired location. Further, we conducted real-time

live-cell imaging within a controlled environment to monitor the outgrowth of neurons while preventing exposure of the neuronal cells to hostile environments. Finite element simulation was

used to guide the appropriate design parameters and verify the two-dimensional model of the proposed structures. Following fabrication of the device, we demonstrated the capability to trap

individual neurons on specific target electrodes with our DEP device. Changes in cell morphology, such as neurite outgrowth, were accurately observed through the transparent substrate in

phase contrast, while avoiding photodamage to neurons that often accompanies fluorescent imaging. Importantly, the applied electric field for DEP did not adversely affect cell health, as

demonstrated by live-cell imaging and immunolabelled images as presented in Fig. 3a,b. Additionally, the accessibility of neurons appropriately grown on the MEAs makes it possible to record

the electrical activity of multiple neurons at the same time, and to investigate the electrical communication between them. Therefore, DEP using MEAs allows not only a label-free and

non-destructive technique for cell manipulation but also a non-invasive interface with biological cells for long-term (days to weeks) monitoring of electrophysiological parameters21,29,30.

Thus, the proposed advanced platform has great potential to form an in vitro ordered neuronal network and allows novel and detailed studies of cellular physiology.

fully-transparent microfluidic DEP device

The proposed fully-transparent microfluidic DEP device is composed of the MEAs integrated glass substrate and microfluidic chip31. The fabrication process is described with schematic

illustrations in Fig. 4a. The fabrication began with ITO deposition and patterning. A 250 nm thick ITO film with a sheet resistance of 6 Ω/□ was deposited on a clean glass substrate by radio

frequency (RF) magnetron sputtering at room temperature. Then, a photolithography and wet-etching processes using hydrochloric acid (HCl) and buffered oxide etchant (BOE) solution were

carried out to define the ring-shaped trap electrode and counter electrodes. A bilayer of Ti (20 nm)/Au (200 nm) was deposited using an electron beam evaporator to serve as the pad electrode

for making a connection between the DEP device and the signal generator (Fig. 4a). Then, a dielectric insulator (SiO2) with a thickness of 200 nm was deposited by an electron-beam

evaporator and patterned by the wet-etching process to define the pad electrodes. The internal diameter and the width of the ring-shaped electrode were 40 μm and 20 μm, respectively. The gap

between the ring electrode and the ground plane was 20 μm. Other parts of the device, such as the contact pads and traces, were made of metal (Ti/Au). The PDMS-based microfluidic chip was

fabricated on a silicon wafer following a previously described soft lithography protocol32. The master replica for rapid prototyping of the PDMS microstructure was patterned using negative

photoresist (SU-8 50, MicroChem Co., Newton, MA) on a silicon wafer. First, a layer of SU-8 was spin-coated at 4000 rpm. The SU-8 coated wafer was baked and exposed through a photomask

containing the desired patterns. After the post-baking treatment, the SU-8 coated wafer was developed leaving master patterns. Liquid PDMS was poured onto the master replica and cured. And

then peeled off the cured PDMS from the master replica after 24 hours. The fabricated PDMS chip was oxygen plasma treated and bonded with the target substrate in which MEAs were fabricated

to form the microfluidic channel (Fig. 4b). The diameters of inlet and outlet holes in the microfluidic chip were 2 mm. The microfluidic channel height, width, and length were 40 μm, 250 μm,

and 3 cm, respectively. Before cells were injected into the microfluidic channel, the fabricated device was cleaned by 75% ethanol and distilled water, and then sterilized by autoclave.

After the autoclaving process, all subsequent procedures were performed in a sterile environment. PDL is commonly coated on tissue cultureware to promote surface adhesion to the cell

membrane. If the channel was immersed in the PDL solution first, the solution would have hindered at the entrance of the channel due to surface tension18,21. Hence, the device was first

treated with 95% ethanol for 5 min, followed by rinsing five times with sterile deionized water. Finally, the inside of the microfluidic channel was coated with a PDL (concentration of 0.1 

mg/

mL).

Fully transparent microfluidic DEP device. (a) Schematic illustration of the fabrication process of the microfluidic DEP device: ITO patterned to form neuron trapping electrodes. Metal

patterning of traces and pads on ITO patterned glass. Electrodes are insulated with SiO2 except metal PADs. Alignment and bonding between electrode patterned substrate and the PDMS

microfluidic chip. (b) Image of the fabricated microfluidic DEP device and optical microscope image of the electrode arrays. Each ring-shaped electrode is surrounded by the reference

electrode and connected to the metal pads to apply AC signals.

The DEP device consists of electrode arrays patterned on a glass slide and PDMS microfluidic chip fabricated using standard photolithography and soft lithography processes as shown in Fig. 

4a. The device features a total of eight trap electrodes located in the center of the device with the ability to control each electrode independently to trap and release cells. The use of

metal (Ti/Au) pads ensures a stable mechanical connection to the cable connectors used for applying the AC signal. Figure 4b shows an image of the fabricated microfluidic DEP device after

the PDMS chip was bonded to the MEAs.

A schematic of the overall experimental set-up for single-neuronal cell trap and culture is depicted in Fig. 5a. The fabricated microfluidic DEP device was placed in the incubator that

incorporates a motorized inverted microscope (BioStation, Nikon, Inc.). and a CCD digital camera (DS-Qi1, Nikon, Inc.) to facilitate live-cell imaging. A mixture of cell culture media and

neurons was loaded onto a 1 mL syringe and the needle inserted into the inlet tube (6.25 × 10−2 inch inner diameter) connected to the microfluidic channel designed to flow the mixture. The

syringe was placed in a syringe pump (Kent Scientific, Genie plus, CT) set a flow rate of 2.5 µL/min. The AC signal used to trap cells on the electrodes was generated by a function generator

(HP 33120 A) and its amplitude and frequency were 8 Vpp and 10 MHz, respectively. In order to prevent signal attenuation, AC signal was applied to the electrode via RF coaxial cable

connectors (Taoglas Limited CAB.058 semi-rigid SMA RF connector), by which impedance was matched to 50 ohms, and was confirmed by the signal measurement using an oscilloscope (Agilent 54621 

A). Operation of the microfluidic DEP device is depicted in Fig. 5b. When the target neuron approaches the trap, the electrode is energized to immobilize the neuron at the center of the

electrode. After neurons are positioned inside the electrodes, a medium without neurons was introduced into the microfluidic channel to remove the excess cells. Neurons attached to the

target electrodes after the medium stopped flowing. The immobilized neurons were cultured and the growth of the neurons was recorded in the incubator.

Single-neuronal cell trapping and culture system. (a) Overview of the cell incubator and monitoring system, as well as an image of the microfluidic DEP device placed in the incubator. The

set-up is composed of a syringe pump, a function generator, an oscilloscope, a camera, a microscope and a monitoring computer. Each metal pad of the eight ring-shaped electrodes is connected

to the positive terminal of the function generator and the two reference electrodes are connected to the ground terminal of the function generator. (b) Schematic illustration of the

cross-section of the microfluidic DEP device.

All animal procedures were approved by the University of Wisconsin Institutional Animal Care and Use Committee (IACUC) and were in accordance with National Institutes of Health guidelines.

Embryonic day (E) 18 cortical/hippocampal neuron cultures were prepared from Sprague-Dawley rats of either sex (Envigo) as described previously33. Briefly, cortices were dissected,

trypsinized and dissociated. Dissociated cortical neurons were plated on 1.0 mg/mL PDL-coated DEP device. Neurons were plated in plating media (PM) (Neurobasal medium with 5% FBS (Hyclone),

B27 supplement, 2 mM glutamine, 37.5 mM NaCl and 0.3% glucose). After 1 h, the medium was replaced with serum-free medium (SFM), which was PM without FBS. Neurons were then fixed and imaged

after 5DIV.

For wide-field imaging, cortical neurons were fixed in 4% paraformaldehyde/Krebs/Sucrose at 37 °C. Cultures were rinsed three times with phosphate buffered saline (PBS) solution and blocked

with 10% BSA/PBS, permeabilized in 0.2% Triton X-100/PBS and labelled with primary and secondary antibodies. Primary antibodies to the α-tubulin, specifically, Tyrosinated-Tubulin

(Millipore) and Tau-1 (Chemicon) and secondary antibodies to goat anti-rat and goat anti-mouse IgG Alexa Fluor 488, 568 and 647 (Invitrogen) were used to visualize microtubules. Phalloidin

coupled to Alexa 488, 568 or 647 (Invitrogen) was used to label actin filaments (1:25 to 1:100). Neurons were imaged on a Nikon TE300 inverted microscope equipped with a 40X/1.3NA Plan Apo

(DIC-fluor) and 20X/0.5NA (phase-fluor) objective. Images were captured on a Coolsnap EZ cooled interline CCD camera (Photometrics).

The data supporting the findings of this study are included within the paper and its Supplementary Information, or available from the corresponding authors upon reasonable request.

References Konry, T., Sarkar, S., Sabhachandani, P. & Cohen, N. Innovative Tools and Technology for Analysis of Single Cells and Cell-Cell Interaction. Annu. Rev. Biomed. Eng. 18, 259–284

(2016).

Article  PubMed  CAS  Google Scholar 

Armbrecht, L. & Dittrich, P. S. Recent Advances in the Analysis of Single Cells. Anal. Chem. 89, 2–21 (2017).

Article  PubMed  CAS  Google Scholar 

Marshel, J. H., Mori, T., Nielsen, K. J. & Callaway, E. M. Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron 67, 562–574 (2010).

Article  PubMed  PubMed Central  CAS  Google Scholar 

Gross, A. et al. Technologies for Single-Cell Isolation. Int. J. Mol. Sci. 16, 16897–16919 (2015).

Article  PubMed  PubMed Central  CAS  Google Scholar 

Pohl, H. A. & Hawk, I. Separation of Living and Dead Cells by Dielectrophoresis. Science 152, 647–649 (1966).

Article  ADS  PubMed  CAS  Google Scholar 

Lim, J. et al. Magnetophoresis of Nanoparticles. ACS Nano 5, 217–226 (2011).

Article  PubMed  CAS  Google Scholar 

Rosenthal, A. & Voldman, J. Dielectrophoretic traps for single-particle patterning. Biophys. J. 88, 2193–2205 (2005).

Article  PubMed  CAS  Google Scholar 

Lan, K. C. & Jang, L. S. Integration of single-cell trapping and impedance measurement utilizing microwell electrodes. Biosens. Bioelectron. 26, 2025–2031 (2011).

Article  PubMed  CAS  Google Scholar 

Bocchi, M. et al. Dielectrophoretic trapping in microwells for manipulation of single cells and small aggregates of particles. Biosens. Bioelectron. 24, 1177–1183 (2009).

Article  PubMed  CAS  Google Scholar 

Schnelle, T. et al. Adhesion-Inhibited Surfaces. Coated and Uncoated Interdigitated Electrode Arrays in the Micrometer and Submicrometer Range. Langmuir 12, 801–809 (1996).

Article  CAS  Google Scholar 

Mittal, N., Rosenthal, A. & Voldman, J. nDEP microwells for single-cell patterning in physiological media. Lab Chip 7, 1146–1153 (2007).

Article  PubMed  CAS  Google Scholar 

Jones, T. B. Electromechanics of particles. (Cambridge University Press, 1995).

Pethig, R. Review article-dielectrophoresis: status of the theory, technology, and applications. Biomicrofluidics 4, 022811 (2010).

Article  PubMed  PubMed Central  CAS  Google Scholar 

Gray, D. S., Tan, J. L., Voldman, J. & Chen, C. S. Dielectrophoretic registration of living cells to a microelectrode array. Biosens. Bioelectron. 19, 771–780 (2004).

Article  PubMed  CAS  Google Scholar 

Khoshmanesh, K., Nahavandi, S., Baratchi, S., Mitchell, A. & Kalantar-zadeh, K. Dielectrophoretic platforms for bio-microfluidic systems. Biosens. Bioelectron. 26, 1800–1814 (2011).

Article  PubMed  CAS  Google Scholar 

Pohl, H. A., Pollock, K. & Crane, J. S. Dielectrophoretic force: A comparison of theory and experiment. J. Biol. Phys. 6, 133–160 (1978).

Article  Google Scholar 

Gross, G. W., Rieske, E., Kreutzberg, G. W. & Meyer, A. A new fixed-array multi-microelectrode system designed for long-term monitoring of extracellular single unit neuronal activity in

vitro. Neurosci. Lett. 6, 101–105 (1977).

Article  PubMed  CAS  Google Scholar 

Jaber, F. T., Labeed, F. H. & Hughes, M. P. Action potential recording from dielectrophoretically positioned neurons inside micro-wells of a planar microelectrode array. J. Neurosci. Methods

182, 225–235 (2009).

Article  PubMed  Google Scholar 

Heida, T., Rutten, W. L. C. & Marani, E. Dielectrophoretic trapping of dissociated fetal cortical rat neurons. IEEE Trans. Biomed. Eng. 48, 921–930 (2001).

Article  PubMed  CAS  Google Scholar 

Yu, Z. et al. Negative Dielectrophoretic Force Assisted Construction of Ordered Neuronal Networks on Cell Positioning Bioelectronic Chips. Biomed. Microdevices 6, 311–324 (2004).

Article  PubMed  CAS  Google Scholar 

Maher, M., Pine, J., Wright, J. & Tai, Y.-C. The neurochip: a new multielectrode device for stimulating and recording from cultured neurons. J. Neurosci. Methods 87, 45–56 (1999).

Article  PubMed  CAS  Google Scholar 

Pohl, H. A. Dielectrophoresis: the behavior of neutral matter in nonuniform electric fields. (Cambridge University Press, 1978).

Morgan, H. & Green, N. G. AC Electrokinetics: Colloids and Nanoparticles. (Research Studies Press, 2003).

Kaler, K. V. & Jones, T. B. Dielectrophoretic spectra of single cells determined by feedback-controlled levitation. Biophys. J. 57, 173–182 (1990).

Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

Pinto, T. M., Wedemann, R. S. & Cortez, C. M. Modeling the electric potential across neuronal membranes: the effect of fixed charges on spinal ganglion neurons and neuroblastoma cells. PLoS

One 9, e96194 (2014).

Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

Zhou, T. et al. Separation and assisted patterning of hippocampal neurons from glial cells using positive dielectrophoresis. Biomed. Microdevices 17, 62 (2015).

Article  CAS  Google Scholar 

De Lima, A. D., Merten, M. D. & Voigt, T. Neuritic differentiation and synaptogenesis in serum-free neuronal cultures of the rat cerebral cortex. J. Comp. Neurol. 382, 230–246 (1997).

Article  PubMed  Google Scholar 

Govek, E.-E., Newey, S. E. & Van Aelst, L. The role of the Rho GTPases in neuronal development. Genes Dev. 19, 1–49 (2005).

Article  PubMed  CAS  Google Scholar 

Wang, X. B., Huang, Y., Wang, X., Becker, F. F. & Gascoyne, P. R. Dielectrophoretic manipulation of cells with spiral electrodes. Biophys. J. 72, 1887–1899 (1997).

Article  PubMed  PubMed Central  CAS  Google Scholar 

Spira, M. E. & Hai, A. Multi-electrode array technologies for neuroscience and cardiology. Nat. Nanotechnol. 8, 83–94 (2013).

Article  ADS  PubMed  CAS  Google Scholar 

Xia, Y. & Whitesides, G. M. Soft Lithography. Angew. Chem. Int. Ed. 37, 550–575 (1998).

Article  CAS  Google Scholar 

Duffy, D. C., McDonald, J. C., Schueller, O. J. A. & Whitesides, G. M. Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). Anal. Chem. 70, 4974–4984 (1998).

Article  PubMed  CAS  Google Scholar 

Viesselmann, C., Ballweg, J., Lumbard, D. & Dent, E. W. Nucleofection and primary culture of embryonic mouse hippocampal and cortical neurons. J. Vis. Exp. 47, e2373 (2011).

Google Scholar 

Download references

This work was supported by Army Research Office (ARO) under grant W911NF-14-1-0652. The program manager is Dr. James Harvey (Dr. Joe X. Qiu, the former). This study was also supported by NIH

grant R01-NS080928 to E.W.D.

Author informationAuthors and Affiliations Department of Electrical and Computer Engineering, University of Wisconsin–Madison, Madison, WI, 53706, USA

Hyungsoo Kim, In-Kyu Lee, Jae Ha Ryu, Sang June Cho, Yei Hwan Jung, Dong-Wook Park, Jihye Bong, Robert H. Blick & Zhenqiang Ma

Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI, 53705, USA

Kendra Taylor

Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, 53706, USA

Karl Richters & Erik W. Dent

Department of Biomedical Engineering, University of Wisconsin–Madison, Madison, WI, 53706, USA

Dong-Hyun Baek, Joseph Novello, Aaron J. Suminski & Justin C. Williams

School of Electrical and Computer Engineering, University of Seoul, Seoul, 02504, South Korea

Dong-Wook Park

Department of Surgery, University of Wisconsin-Madison, Madison, WI, 53706, USA

Aaron M. Dingle

AuthorsHyungsoo KimView author publications You can also search for this author inPubMed Google Scholar

In-Kyu LeeView author publications You can also search for this author inPubMed Google Scholar

Kendra TaylorView author publications You can also search for this author inPubMed Google Scholar

Karl RichtersView author publications You can also search for this author inPubMed Google Scholar

Dong-Hyun BaekView author publications You can also search for this author inPubMed Google Scholar

Jae Ha RyuView author publications You can also search for this author inPubMed Google Scholar

Sang June ChoView author publications You can also search for this author inPubMed Google Scholar

Yei Hwan JungView author publications You can also search for this author inPubMed Google Scholar

Dong-Wook ParkView author publications You can also search for this author inPubMed Google Scholar

Joseph NovelloView author publications You can also search for this author inPubMed Google Scholar

Jihye BongView author publications You can also search for this author inPubMed Google Scholar

Aaron J. SuminskiView author publications You can also search for this author inPubMed Google Scholar

Aaron M. DingleView author publications You can also search for this author inPubMed Google Scholar

Robert H. BlickView author publications You can also search for this author inPubMed Google Scholar

Justin C. WilliamsView author publications You can also search for this author inPubMed Google Scholar

Erik W. DentView author publications You can also search for this author inPubMed Google Scholar

Zhenqiang MaView author publications You can also search for this author inPubMed Google Scholar

H.K. and Z.M. designed the experiments. H.K., I.K.L. and J.H.R., carried out the device fabrication. H.K., I.K.L., K.T., K.R., D.H.B., J.H.R., S.J.C., Y.H.J., D.P., J.N., J.B., R.H.B.,

J.C.W., E.W.D. and Z.M. carried out the experimental validation and analysis. H.K., I.K.L., K.T., A.J.S., A.M.D., J.C.W., E.W.D. and Z.M. wrote the paper. All images and figures were taken

and drawn by H.K. and I.K.L.

Corresponding authors Correspondence to Justin C. Williams, Erik W. Dent or Zhenqiang Ma.

The authors declare no competing interests.

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About this articleCite this article Kim, H., Lee, IK., Taylor, K. et al. Single-neuronal cell culture and monitoring platform using a fully transparent microfluidic DEP device. Sci Rep 8,

13194 (2018). https://doi.org/10.1038/s41598-018-31576-2

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Received: 20 March 2018

Accepted: 15 August 2018

Published: 04 September 2018

DOI: https://doi.org/10.1038/s41598-018-31576-2

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