Efficient Dielectrophoretic Patterning of Embryonic Stem Cells
in Energy Landscapes Defined by Hydrogel Geometries
HIDEAKI TSUTSUI,1EDMOND YU,1SABRINA MARQUINA,1BAHRAM VALAMEHR,2IEONG WONG,1
HONG WU,2and CHIH-MING HO1
1Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA;
and2Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA
(Received 21 January 2010; accepted 18 June 2010; published online 8 July 2010)
Associate Editor Michael S. Detamore oversaw the review of this article.
Abstract—In this study, we have developed an integrated
microfluidic platform for actively patterning mammalian
cells, where poly(ethylene glycol) (PEG) hydrogels play two
important roles as a non-fouling layer and a dielectric
structure. The developed system has an embedded array of
PEG microwells fabricated on a planar indium tin oxide
(ITO) electrode. Due to its dielectric properties, the PEG
microwells define electrical energy landscapes, effectively
forming positive dielectrophoresis (DEP) traps in a low-
conductivity environment. Distribution of DEP forces on a
model cell was first estimated by computationally solving
quasi-electrostatic Maxwell’s equations, followed by an
experimental demonstration of cell and particle patterning
without an external flow. Furthermore, efficient patterning of
mouse embryonic stem (mES) cells was successfully achieved
in combination with an external flow. With a seeding density
of 107cells/mL and a flow rate of 3 lL/min, trapping of cells
in the microwells was completed in tens of seconds after
initiation of the DEP operation. Captured cells subsequently
formed viable and homogeneous monolayer patterns. This
simple approach could provide an efficient strategy for
fabricating various cell microarrays for applications such as
cell-based biosensors, drug discovery, and cell microenviron-
Keywords—Poly(ethylene glycol), Surface engineering, Cell
microarray, Microfluidics, Dielectrophoresis.
Surface-patterned cell arrays are indispensable
platforms for cell-based sensors,5,12drug discovery,9
as well as investigating cell–cell or cell–matrix inter-
actions in a well-controlled manner in vitro.13,41
Advances in surface chemistry and material sciences in
conjunction with developments of micro/nano fabri-
cation techniques have led to various methods of cel-
lular patterning,12,13,32,44including, but not limited to,
majority of such cellular patterning methods rely on
spatially defined so-called non-fouling surfaces on a
substrate (e.g., glass, silicon, and polystyrene plate), to
which non-specific binding of protein components
from cell culture media is significantly reduced or
eliminated, compared with unmodified surfaces. A
sequence of coating with cell-adhesive molecules such
as extracellular matrix (ECM) proteins, charged poly-
mers, or antibodies, washing off the adhesive compo-
nents from the non-fouling areas, incubating with
target cells, and carefully rinsing to remove excess cells
leads to defined patterns of the cells on a fabricated
substrate. Such surface-based cell patterning is passive
in nature and has several drawbacks; (a) settlement of
cells takes a few hours, (b) falling of cells is a stochastic
process, leading to non-uniform cell distributions, (c)
the majority of cells falling onto non-fouling surface
are wasted, and (d) developing clean patterns of cells
usually requires extremely careful washing.
Dielectrophoresis (DEP) is the motion of a polar-
fields,21,30and has been frequently employed in cell
and particle separation/sorting in microfluidic sys-
tems.29,40Recently, DEP forces generated by a pat-
terned microelectrode array were used to actively
pattern bovine pulmonary arterial endothelial cells
on the trapping electrodes.16In this study, the DEP
Address correspondence to Chih-Ming Ho, Department of
Angeles, Los Angeles, CA 90095, USA. Electronic mail: chihming@
Annals of Biomedical Engineering, Vol. 38, No. 12, December 2010 (? 2010) pp. 3777–3788
0090-6964/10/1200-3777/0 ? 2010 The Author(s). This article is published with open access at Springerlink.com
patterning demonstrated significantly more accurate
patterning of cells on desired spots compared with
poor patterning via conventional surface modification
methods, while not compromising viability and growth
of the patterned cells. In other studies, a layer of an
electrically insulating photoresist was patterned on a
planar electrode to create a virtual electrode array for
temporal cell trapping and subsequent encapsulation
in a hydrogel.1,2As demonstrated in these studies,
insulating materials can be used to shape highly non-
uniform electric fields required for DEP manipulation
(insulator-based DEP) of cells and particles,11while
significantly simplifying the fabrication process and
improving ease of operations. However, commonly
used insulating polymer materials such as SU-8 and
polydimethylsiloxane (PDMS) do not have good non-
fouling characteristics and inefficiently prevent non-
specific binding of protein components as well as cell
attachment and growth. Therefore, it is highly desired
that the insulating layer is made of non-fouling mate-
rials for improved fidelity and long-term stability of the
defined cell patterning. Here we propose to develop a
quick (~tens of seconds) and active cell patterning
method based on positive dielectrophoretic (DEP)
traps, which are defined by non-fouling microwell
The time-averaged DEP force FDEPon a particle of
radius r, imposed by an AC electric field E of angular
frequency x, is given as21,43:
FDEP¼ 2pemr3Re fCM
½ ?r Erms
where emis the permittivity of the surrounding med-
ium, and fCM is the Clausius–Mossoti (CM) factor
particle and the surrounding medium, respectively.
Each complex permittivity takes the form e* = e 2 jr/
x, where e and r are the permittivity and electrical
conductivity of the corresponding substances, respec-
tively. j ¼
the CM factor, Re fCM
relative strength of the DEP force in a given electric
field; when it is positive the particle is pulled toward a
higher electric field region (positive DEP), whereas
when negative the particle is pulled away from a higher
electric field region (negative DEP).
mare the complex permittivities of the
is the imaginary unit. The real part of
?, dictates the direction and
According to Eq. 1 the DEP force is proportional to
magnitude of the electric field is critical for generating
substantial DEP forces. In this study, we use dielectric
microwell structures made of a non-fouling PEG
hydrogel to create such a non-uniform electric field
(Fig. 1). Under current experimental conditions with a
small length scale (~100 lm) and a moderate AC fre-
quency (~10 MHz), quasi-electrostatic estimation of
the time-varying electric field can be applied. There-
fore, we solved quasi-electrostatic Maxwell’s equations
jj2, meaning that non-uniformity but not mere
E ¼ ?r/
r ? J þ@q
r ? D ¼ q
where / is the electric potential, J is the current vector,
q is the free charge density, and D is the electric flux
density. Considering J = rE and D = eE for a
homogeneous linear dielectric substance, Eq. 3 is
combined to result in:
r ? rr/
@tr ? er/
ðÞð Þ ¼ 0:
By introducing a phasor~/ x;t
Eq. 4 reduces to Laplace’s equation for the real and
imaginary parts of the phasor,
ð Þ ¼ /Rþ j/I
Equation 5 can be solved numerically for a model 2-
D space with boundary conditions described in Fig. 2.
The electric field is then calculated from Eq. 6 and used
to estimate available DEP force in Eq. 1.
E ¼ Re ?r~/
¼ Re ?rð/Rþ j/IÞejxt
MATERIALS AND METHODS
Poly(ethylene glycol) diacrylate (PEG-DA, Mn ~
258), 2,2-Dimethoxy-2-phenylacetophenone (DMPA),
3-(Trimethoxysilyl)propyl acrylate (TPA), ethanol,
toluene, triethylamine, HEPES, calcium chloride,
b-mercaptoethanol, and gelatin were purchased from
Sigma-Aldrich (St. Louis,
1,1,2,2-tetrahydrooctyl) trichlorosilane, vinyltrimeth-
oxysilane and 10-undecenyl trimethoxysilane were
purchased from Gelest (Morrisville, PA). Indium tin
TSUTSUI et al.3778
oxide (ITO)-coated glass slides were purchased from
Delta Technologies (Stillwater, MN). PDMS pre-
polymer and curing agent (Sylgard 184) were obtained
from Dow Corning (Midland, MI). SU-8 photoresist
and SU-8 developer were purchased from MicroChem
Eagle’s medium (KO-DMEM), phosphate buffered saline
(PBS), fibronectin, trypsin,
streptomycin, non-essential amino acids (NEAA), and
LIVE/DEAD viability/cytotoxicity kit were purchased
metal electrodes; electrical interaction between a non-uniform electric field and polarized charges induced by the field exerts a net
force on the particle toward a higher electric field region. (b) Dielectric properties of PEG microstructures can be used to generate a
non-uniform electric field required for positive DEP manipulation of mammalian cells. (c) A three-step approach for efficient
patterning of cells. (c-i) Non-fouling property of PEG allows for selective coating of ECM proteins in the microwell. (c-ii) cells are
attracted to the microwells due to positive DEP forces. (c-iii) After removal of electrical potential and gentle washing, excess cells
are removed to reveal a monolayer of patterned cells.
Proposed cell patterning method using positive DEP force defined by non-fouling microstructures. (a) Positive DEP by
and boundary conditions for solving Laplace’s equation (Eq. 5). The microwell’s diameter and thickness are 100 lm and 10 lm,
respectively. V05 10 V. (c) Numerical solution of DEP force exerted on a model mammalian cell, where magnitudes and directions
of the force vectors are represented by a color map and arrows, respectively. Contours represent electrical potential (DV 5 0.5 V).
Finite element analysis of positive DEP force on a mammalian cell. (a, b) Computational domain (1000 lm 3 100 lm)
Efficient Dielectrophoretic Patterning3779
from Invitrogen (Carlsbad, CA). Fetal bovine serum
(FBS) was purchased from HyClone (Logan, UT).
Leukemia inhibitory factor (LIF) was purchased
from Millipore (Temecula, CA). 7-Aminoactinomycin
D (7-AAD) was purchased from BD Biosciences (San
Jose, CA). Polystyrene microspheres (25 lm mean
diameter) were purchased from Duke Scientific (Palo
Finite-Element Method Analysis
A commercially available finite-element solver,
COMSOL Multiphysics (COMSOL, Stockholm, Swe-
den), was used to solve the Laplace’s equation (Eq. 5)
for the electric field modulated by the PEG microwells.
With obtained electric field, theoretical DEP force on a
model mammalian cell was computed from Eq. 1.
Fabrication of PEG Microwells
and Microfluidic Systems
PEG microwells were made on ITO glass slides
using PDMS mold-based soft lithography. The mold
master was fabricated by photolithography of SU-8
photoresist on a silicon wafer. Prior to application of
PDMS pre-polymer, the master was treated with va-
por-phase (tridecafluoro-1,1,2,2-tetrahydrooctyl) tri-
chlorosilane for 15 min in a vacuum desiccator to
prevent bonding between the master and the cured
PDMS mold. 60 g of the PDMS mixture (pre-polymer
10: curing agent 1) was applied on the master and
cured for 2 h at 65 ?C. The PDMS mold was then
peeled from the master and cut into each piece. Inlet
and outlet holes were punched through the mold.
Similar to the mold master, surface of the PDMS mold
PEG microwells were fabricated on an ITO glass
slide which served as the bottom plate of the micro-
fluidic channel as well as the bottom electrode. First,
ITO surface was functionalized with acrylate groups
(Fig. 3a). ITO glass slides were cleaned with solvent
wash (acetone, methanol, and isopropanol), rinsed
with deionized (DI) water, and dried under nitrogen
stream. Cleaned ITO slides were then treated with
oxygen plasma (Tegal Plasma Asher, Tegal, Petaluma,
CA) for 10 min and immediately incubated in a tolu-
ene solution containing 3 mM TPA and 1% triethyl-
amine for 1 h. The modified ITO slides were rinsed
with toluene, isopropanol, and DI water, followed by
nitrogen dry. All slides were stored in a vacuum des-
iccator until use.
To fabricate the PEG microwells, the PDMS mold
was firmly placed on the modified ITO slide, forming a
void space which would define geometries of the PEG
layer (Fig. 3b). PEG-DA pre-polymer containing the
photo initiator DMPA (1% w/w) was introduced to
the void space using capillary force (Fig. 3c). Upon
complete filling of the void space by PEG-DA solution,
the slide was exposed to 1 W/cm2of UV light for 60 s
using a UV spot cure system (EXFO OmniCure S1000,
EXFO, Ontario, Canada) (Fig. 3d). After removal of
the PDMS mold, excess volume of PEG-DA was
trimmed to complete fabrication of PEG hydrogel
microwell layer (Fig. 3e). The PEG microwell chip was
incubated with an ECM protein solution (20 lg/mL
fibronectin in PBS) for 30 min, followed by DI water
rinse and nitrogen dry (Fig. 3f). The coated chip was
typically used within a day.
The top assembly of the microchannel was fabri-
cated by similar molding of PDMS. First, a small piece
of clean ITO slides (approximately 10 mm 9 10 mm)
were modified with 10-undecenyl trimethoxysilane
(3 mM solution in toluene containing 1% triethyl-
amine) following the same protocol as for the TPA
modification. This modification rendered vinyl groups,
allowing for formation of covalent linking to PDMS
microchannel during the curing process. After electri-
cal wiring (i.e., a conductive copper tape) was attached,
this ITO piece was placed on the SU-8 channel mold
master (Fig. 3g), and PDMS pre-polymer was poured
and cured for 2 h at 65 ?C (Fig. 3h). The PDMS
channel was then peeled from the master and cut into
Fabrication of a microfluidic channel with an
TSUTSUI et al.3780
each piece. Inlet and outlet holes were punched
through the mold (Fig. 3i). Finally fabrication of the
microfluidic channel system was completed by assem-
bling the top PDMS microchannel and the bottom
ITO slide with PEG microwells and inserting stainless
steel coupling tubes into the channel inlet and outlet
Cell Culture and Preparation
Murine ES cells LW124were routinely cultured
on irradiated mouse embryonic fibroblast (MEF)
cells in Knock-Out DMEM supplemented with 15%
FBS, 1000 U/mL leukemia inhibitory factor, 2 mM
L-glutamine, penicillin, streptomycin, 0.1 mM non-
essential amino acids, and 100 lM b-mercaptoethanol.
This culture medium is referred as ES medium
throughout this article. ES cells were fed with fresh
medium every other day. Before formation of cell
arrays, ES cells were transferred to gelatin-coated
plates and cultured for two passages to eliminate
residual MEF cells from culture. All cultures were
maintained in a 37 ?C humidified incubator supple-
mented with 5% CO2.
Immediately prior to cell patterning experiments,
ES cells were trypsinized, resuspended in an isoos-
motic, low-conductivity buffer solution (LCB), and
kept on ice. LCB was DI water-based solution con-
taining 10 mM HEPES, 0.1 mM calcium chloride,
59 mM D-glucose, and 236 mM sucrose,3and its pH
was adjusted to 7.35 by NaOHaq, with a final con-
ductivity of 0.020 S/m.
Dielectrophoretic Patterning of ES Cells
The assembled microchannel was mounted on a
Leica DMIRB inverted fluorescence microscope (Leica
Microsystems, Bannockburn, IL) equipped with a
CoolSNAP HQ CCD camera (Photometrics, Tucson,
AZ) for image recoding. During the patterning process
and the following culture period, temperature (37 ?C)
and CO2level (5%) were maintained using a stage-top
incubator system (Incubator L, CTI-Controller 3700,
Tempcontrol 37-2 digital, and Heating Stage, all from
PeCon GmbH, Erbach, Germany).
The microchannel was first fed with ethanol to
remove air bubbles, followed by complete replacement
with LCB. All fluidic flow was operated by a peristaltic
pump (Instech Laboratories, Plymouth Meeting, PA)
controlled by LabVIEW software (National Instru-
ments, Austin, TX). Cell suspension was introduced
into the channel and, when the cells reached the
microwell array section, AC electrical potential (20 Vpp
at 10 MHz) was applied between the electrodes by a
function generator (33120A, Hewlett-Packard, Palo
Alto, CA) to initiate the DEP cell patterning process.
Upon completion of patterning, AC potential was
removed, and LCB was replaced with fresh ES medium
described above. Environmental control (37 ?C tem-
perature and 5% CO2) and continuous feeding of the
medium were continued until experiments were termi-
Cell Viability Assay
Two types for cell viability tests were used in this
study. First, effects of LCB on the viability of mES
cells were investigated by flow cytometry with 7-AAD
staining. Briefly, cells maintained under the standard
culture condition were dissociated into single cells
using trypsin, centrifuged for 5 min, resuspended in
LCB, and kept either on ice or at room temperature
for 30 min. A portion of cells were then transferred to
adhesion culture with the ES medium on a fibronec-
tin-coated multi-well plate for later analyses. The
remaining portion was collected, centrifuged for
5 min, resuspended in 350 lL of Hank’s Buffered Salt
Solution (HBSS) containing 4% serum, incubated
with 7 lL of 7-AAD solution for 5 min, and kept on
ice before FACS analysis. Non-viable cells are readily
stained by 7-AAD, a nucleic acid dye, penetrating
through the compromised cell membrane, while viable
acquired and analyzed with BD FACS Canto II Flow
Cytometry System (BD Biosciences, San Jose, CA).
To determine the gating for non-viable population, a
mixture of viable ES cells and non-viable ES cells,
prepared by incubating with 0.1% saponin in D-PBS
for 10 min, was used to define gating for live cell
population as shown in Fig. 4. The mES cells trans-
ferred to and maintained on the adhesion culture
were similarly analyzed after 24 h and 72 h counting
from the 30-min incubation in LCB.
Viability of cells after patterning was measured
using Invitrogen’s LIVE/DEAD viability/cytotoxicity
kit (Cat. No. L3224) by following supplied instruc-
tions. Briefly, the patterned cells were incubated for
30 min in 5 lM calcein AM and 5 lM ethidium
homodimer-1 (EthD-1) in D-PBS. Calcein AM is a
cell-permeant dye that is enzymatically converted to
the intensely green fluorescent calcein in viable cells,
while EthD-1 can only enter cells through compro-
mised membrane of non-viable cells and stain red after
binding to the nucleic acids. Fluorescence images of
the stained cells were captured using the inverted
Efficient Dielectrophoretic Patterning3781
RESULTS AND DISCUSSIONS
Finite-Element Method Analysis
dielectric constant of PEG-DA is approximately 10 in
the frequency range between 1 Hz and 10 MHz. Con-
ductivity of PEG-DA was also estimated from experi-
mental data available in the literature.4,10However, the
conductivity of PEG-DA was not necessary for the
current analysis as either of the Laplace’s equations nor
the CM factor required this value. The electrical
properties of the LCB and mammalian cells were
adapted from the literature.2,15List of materials’ con-
ductivity and permittivity are summarized in Table 1.
The estimated DEP force field on the model cell
around a PEG microwell (100 lm diameter and 10 lm
thickness) is shown in Fig. 2c. Distribution of the DEP
to published experimentalwork,31
force, where arrows and the color map indicate direc-
tion and magnitude of the force, respectively, predicted
movement of all viable cells into the microwell. It was
also anticipated that the cells will be primarily
attracted to the edges where maximum DEP force is
trypsinized, centrifuged, resuspended in LCB, and kept for 30 min either on ice or at room temperature (RT). Cells were then
transferred to adhesion culture on fibronectin-coated plates supplemented with the ES medium, and maintained in a tissue culture
incubator. Cells were sampled immediately after the 30-min LCB incubation and 24 h and 72 h thereafter, and analyzed for viability
using 7-AAD staining. An equal mixture of healthy and dead mES cells was used to define a gating for the live population (‘‘Gating
Control’’). Routinely maintained mES cells without LCB incubation were used as a positive control (‘‘ES Medium’’). Based on
percentage of viable cells, no adverse effects on cell viability due to 30-min LCB incubation were observed. FSC-A: forward
scattering area; PerCP-Cy5-5-A: light intensity corresponding to 7-AAD’s emission.
Flow cytometry analysis of mES cell viability after a 30-min incubation in LCB. Routinely cultured mES cells were
TABLE 1. Electricalproperties
Dielectric constant e
(e0= 8.854 9 10212Fm21)
3 9 1026
TSUTSUI et al.3782
Viability Tests of Cells Incubated in LCB
Positive DEP manipulation of the cells requires a
low-conductivity environment. We thus use LCB while
patterning the cells and later replace it with the ES
medium. In our protocol, mES cells are typically
exposed to LCB for less than 30 min. To evaluate
effects of LCB on the viability of cells, we first incu-
bated mES cells in LCB for 30 min and measured the
viability using 7-AAD staining and flow cytometry at
three different time points: immediately after the
incubation (0 h), after 24-h and 72-h incubations in the
ES medium. As shown in Fig. 4, 30-min incubations in
LCB either on ice or at room temperature (RT) did not
compromise apparent cell viability. This result indi-
cates that the necessary exposure to LCB during DEP
patterning has minimal effects on the immediate and
subsequent cell viability.
Cell Patterning without an External Flow
We first demonstrated patterning of polystyrene
beads (negative DEP particles) and mES cells (positive
DEP particles) without an external flow. Once particles
were introduced to the microwell array section, the
flow was stopped and DEP patterning was initiated by
applying an AC potential (20 Vpp at 10 MHz). As
shown in Fig. 5, 92% of mES cells were trapped in the
microwells within 10 s, while 88% of polystyrene beads
were pushed away from the wells by negative DEP
force, forming a rectangular grid pattern which cor-
responded to local minima of the electric field strength.
The 8% of mES cells remained outside of the wells are
likely due to spontaneous adhesion of the cells to the
substrate. On the other hand, it seems that 12% of the
beads remained inside the wells because of the local
minimum of the electric field strength at the center of
the wells. This set of experiments proved that electric
field was indeed modulated by the PEG microwells,
forming positive DEP trap arrays as designed.
Cell Patterning with an External Flow
After successful entrapment of mES cells without an
external flow, we demonstrated DEP patterning of
mES cells in a continuous flow (flow rate: 3 lL/min)
with two different cell seeding density: 106cells/mL
low-conductivity environments. (a) Polystyrene beads (25 lm) and mES cells demonstrated opposite movements; the polystyrene
beads experienced negative DEP forces and the majority moved to regions of weak electric field, while almost all mES cells
experiencing positive DEP forces were trapped in the microwells where electric field is strong. Scale bars: 200 lm. (b) Frequency of
the polystyrene beads and mES cells found inside and outside of the microwells before and after DEP manipulations. For each
sample, the error bar indicates the standard deviation calculated from three data sets.
DEP manipulation of polystyrene beads (negative DEP) and mES cells (positive DEP) without an external flow under
Efficient Dielectrophoretic Patterning3783
and 107cells/mL. At the lower cell seeding density,
majority of cells in the field of view immediately
responded to the applied electric field and moved to the
microwells within 1 s after initiation of DEP manipu-
lation (Fig. 6a). On the other hand, due to a small
number of incoming cells, filling of the microwells
required a longer time. At a higher cell seeding density,
initial response of individual cells was relatively slow.
Nevertheless, a larger number of incoming cells
allowed for almost complete filling of the microwells
within 25 s (Fig. 6b).
In another set of experiments, continuous operation
for a period of a few minutes allowed for complete
filling of the microwells with mES cells and subsequent
formation of uniform monolayer patterns upon
removal of the AC potential and a gentle rinsing step.
After replacement of LCB with the fresh ES medium
and 1 day of continuous culture, a high viability of the
patterned cells (>95% live) was confirmed by the live/
dead staining assay (Fig. 6c). This positive DEP-based
patterning approach requires cells to be exposed to
environments that are not common for routine tissue
culture, i.e., a low-conductivity medium and a highly
non-uniform electric field. Thus, typical patterning
operations are completed in a relatively short time so
that cells are only exposed to LCB for at most 30 min
and the electric field for 3 min or less. While this
strategy allows for survival of most mES cells during
the patterning process, long-term effects on cell growth
and differentiation need to be investigated in future
Device Development and Operations
In order to develop the proposed microfluidic
device, various key fabrication techniques were adap-
ted or developed. The most critical one among them
was to engineer interfaces so that secure covalent
formation of a viable homogeneous cell array. (a) At a lower cell seeding density (106cells/mL), majority of cells in the field of view
were trapped in the wells within 1 s after initiation of DEP manipulation. Due to smaller number of incoming cells, filling of
microwells required a longer time. Applied AC potential was 20Vppat 10 MHz. Flow direction and rate were from left to right and
3 lL/min (approximately an average speed of 250 lm/s), respectively. (b) At a higher cell seeding density (107cells/mL), initial
response of individual cells were relatively slow. Nevertheless, a larger number of incoming cells allowed for almost complete
filling of microwells within 25 s. Performed under the same experimental conditions as (a) except for the cell seeding density.
(c) Bright field and fluorescent images of the patterned mES cells stained using the LIVE/DEAD kit after 1 day from the DEP
patterning operation. Scale bars: 200 lm (a, b), 100 lm (c), and 50 lm (insets of c), respectively.
DEP patterning of mES cells with an external flow, demonstrating quick capture and assembly of the cells and
TSUTSUI et al.3784
bonds were formed between ITO and PEG-DA as well
as between glass and PDMS. On glass or silicon sub-
strates, PEG-DA hydrogel is typically immobilized
by silane modification of surface hydroxyl groups
with acrylate-terminated trichloro- or trimethoxy-silane
(e.g., TPA) and subsequent free radical polymerization
of PEG-DA pre-polymers.22,27,33,37While this is a
robust approach on glass and silicon substrates, very
few reported the same modification strategy on ITO.
In our study, only the trimethoxy silane (i.e., TPA)
successfully rendered reliable covalent linking on ITO.
3-(Trichlorosilyl)propyl acrylate, on the other hand,
noticeably eroded the ITO surface and did not yield
stable coupling with PEG-DA hydrogel. The other key
development was functionalization of the top electrode
ITO glass piece with vinyl group so that covalent link
would be formed between the top electrode and PDMS
channel during the curing process of PDMS (Fig. 3h).
We tested two trimethoxysilane molecules with a vinyl
(C14H30O3Si) and vinyltrimethoxysilane (C5H12O3Si),
along with controls (glass and ITO with or without
plasma treatment) and found that only the substrates
treated with 10-undecenyltrimethoxysilane successfully
yielded stable adhesion between PDMS and both ITO
and glass. Although detailed investigation is required,
it is likely that the long carbon chains of this silane
molecule improved chances of polymerization between
two vinyl groups from the silane monolayer on ITO
surface and the PDMS pre-polymer. To our knowl-
edge, this is the first demonstration of a PDMS chan-
nel with a literally embedded ITO glass electrode. Due
to ease of both fabrication and subsequent assembly
with a bottom electrode plate, this technique could
provide a convenient way of fabricating microfluidic
systems with top and bottom parallel electrodes.3,8
In this study, we found that available DEP force
significantly changed within the frequency range of
1–10 MHz (Fig. 7a). This frequency range is widely
used for positive DEP manipulation of cells by metal
electrode-based devices because the CM factor peaks
within this range, offering the maximum DEP force
(Fig. 7b). However, an insulator-based electrode array
in the current system is more sensitive to the operation
mES cells was observed at frequencies below 10 MHz. Seeding density: 106cells/mL and flow rate: 3 lL/min. (b) Frequency
dependence of the CM factor having a wide peak band between 1 MHz and 10 MHz. (c) Interfacial charge accumulation and
corresponding potential drop (DVm) across the medium phase (i.e., LCB) was estimated by using a simple one-dimensional model
(see inset). This model indicates that the interfacial charges are no longer negligible below 10 MHz and causes significant loss of
DVm, meaning non-uniform electric fields required for DEP manipulation are equally attenuated. Scale bars: 200 lm.
Frequency dependence of DEP force and patterning efficiency. (a) Significant reduction of positive DEP forces on
Efficient Dielectrophoretic Patterning3785
frequency due to non-negligible charge accumulation
at the interface between the solution and PEG and
subsequent potential drop across the PEG layer at
frequencies below 10 MHz (Fig. 7c, see Appendix).
Therefore, this device must be operated at ~10 MHz in
order to maximize contributions from both the CM
factor and r Erms
jj2in Eq. 1.
Hydrogel Materials Selection
In this study, PEG-DA 258 (Mn ~ 258) was chosen
as the building material of the microwells. It is known
that the non-fouling property of PEG-based hydrogels
improves with the molecular weight (MW) of the pre-
polymer PEG-DA. In a recent extensive study by
Moeller et al.,27microwells made of PEG-DA 258
suffered from significant protein absorption and cell
adhesion compared with microwells made of larger
MW PEG-DAs such PEG-DA 575 (Mn ~ 575) and
PEG-DA 1000 (Mn ~ 1000). This relatively low non-
fouling property led to outgrowth of embryoid bodies
(EBs) and poor retrieval from the microwells. On the
other hand, hydrogels made of higher MW PEGs
undergo significant swelling under aqueous condi-
tions,26leading to poor structural stability of the
microwells anchored on substrates.27Consequently,
pre-polymer solutions of the above-mentioned PEG-
DA 575 and 1000 needed to be significantly diluted
(e.g., 20% PEG-DA: 80% PBS) so that relative
amount of swelling upon full hydration was small
enough.27In this study, in addition to providing non-
fouling surfaces and physical structures, PEG micro-
wells play a key role as a dielectric component, creating
a strong non-uniform electric field for actively trapping
mES cells. Hence, the use of diluted (or pre-hydrated)
high MW PEG-DA which lowers the relative dielectric
constant of the microwells is not desirable. Conse-
quently, PEG-DA 258 seems to be the best compro-
mise for the proposed application at this moment.
Beyond PEG-based hydrogels, more robust polymer
materials such as polyacrylamide28
recently demonstrated excellent non-fouling proper-
ties, and they might serve as an excellent substitute
material for the current application in the future.
We have developed a novel microfluidic platform
with an embedded array of PEG-DA microwells for
actively patterning mES cells by positive DEP. The
uniqueness of our approach lies in the use of surface-
engineered PEG-DA hydrogel not only as a typical
non-fouling layer, but also as a key electrical compo-
nent to define electrical energy landscapes within a
microfluidic system. Due to dielectric properties of the
PEG microwells, an applied AC potential by ITO
electrodes creates a strong non-uniform electric field
around the microwells, forming positive DEP traps for
mammalian cells in a low-conductivity environment.
allowed fast trapping and patterning of mES cells in
the microwells within tens of seconds. Captured cells
subsequently formed homogeneous monolayer and
remained mostly viable as confirmed by the live/dead
assay. Furthermore, as evidenced in Figs. 5 and 6, the
positive DEP traps successfully collected the majority
of the cells available in the field. Therefore, total
consumption of cells could be minimized with this
approach when compared to conventional surface-
based patterning methods where a large number of
cells are required and eventually wasted. In addition,
this efficient patterning method can be easily applied to
the patterning of other types of mammalian cells. Due
to the simplicity of the device design and operation,
this technology is readily scalable and could facilitate
manufacturing of large-scale cell arrays.
CM Factor Calculation
In order to calculate the CM factor in Eq. 2, a
mammalian cell was modeled as a spherical concentric
dielectric shell with an outer radius r (=4.0 9 1026m)
having shell permittivity e?
core permittivity e?
(=8.0 9 1029m)
mem(=emem2 jrmem/x) and
The CM factor was then calculated from Eqs. 2
and 7 using MATLAB (MathWorks, Matick, MA).
Parameter values used were adapted from the published
literature15and listed in Table 1.
Interfacial Charge Accumulation
Positive DEP manipulation of mammalian cells in a
low-conductivity environment is typically operated at a
frequency range 1–10 MHz where the CM factor
peaks, maximizing available DEP force. Nevertheless,
in this experimental study, significant loss of attractive
force was observed at frequencies below 10 MHz. This
is because the assigned interfacial condition in Fig. 2
(i.e., no charge accumulation at interface between the
TSUTSUI et al.3786
PEG-DA and LCB) is no longer valid at these fre-
quencies. At a lower frequency, charges can transfer
fast enough to accumulate at the interface, counter-
acting the electric field generated by the applied AC
potential. In other words, accumulated charges effec-
tively reduce potential drop across the fluid domain
and subsequently minimize magnitude of non-uniform
electric field required for DEP forces (see Eq. 1).
We attempted to estimate the interfacial charge
accumulation qsuby assuming a simple model system
shown in the inset of Fig. 7c and using a corresponding
where r, e, and h are conductivity, permittivity, and
height, respectively, and subscripts a and b indicate
Þ þ jx hbeaþ haeb
This study is supported by the Center for Cell
Control (1PN2 ey018228) through the NIH Roadmap
for Nanomedicine, and the Center for Scalable
and Integrated Nanomanufacturing (SINAM) under
National Science Foundation (CMMI-0751621).
This article is distributed under the terms of
the Creative Commons Attribution Noncommercial
License which permits any noncommercial use, distri-
bution, and reproduction in any medium, provided the
original author(s) and source are credited.
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