Efficient Dielectrophoretic Patterning of Embryonic Stem Cells in Energy Landscapes Defined by Hydrogel Geometries
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 10⁷ cells/mL and a flow rate of 3 μL/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 microenvironment studies.
Efﬁcient Dielectrophoretic Patterning of Embryonic Stem Cells
in Energy Landscapes Deﬁned by Hydrogel Geometries
and CHIH-MING HO
Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA;
Department 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
cells/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,
as well as investigating cell–cell or cell–matrix inter-
actions in a well-controlled manner in vitro.
Advances in surface chemistry and material sciences in
conjunction with developments of micro/nano fabri-
cation techniques have led to various methods of cel-
including, but not limited to,
and inkjet printing.
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-
izable particle induced by non-uniform electric
and has been frequently employed in cell
and particle separation/sorting in microﬂuidic sys-
Recently, DEP forces generated by a pat-
terned microelectrode array were used to actively
pattern bovine pulmonary arterial endothelial cell s
on the trapping electrodes.
In this study, the DEP
Address correspondence to Chih-Ming Ho, Department of
Mechanical and Aerospace Engineering, University of California, Los
Angeles, Los Angeles, CA 90095, USA. Electronic mail: chihming@
Annals of Biomedical Engineering, Vol. 38, No. 12, Decem ber 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 hydro gel.
As 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,
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
structures made of poly(ethylene glycol) (PEG)
The time-averaged DEP force F
on a particle of
radius r, imposed by an AC electric ﬁeld E of angular
frequency x, is given as
is the permittivity of the surrounding med-
ium, and f
is the Clausius–M ossoti (CM) factor
are the complex permittivities of the
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
is the imaginary unit. The real part of
the CM factor, Re f
½, dictates the direction and
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).
According to Eq. 1 the DEP force is proportional to
, meaning that non-uniformity but not mere
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 exp erimental 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. Ther e-
fore, we solved quasi-electrostatic Maxwell’s equations
rD 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 er/ðÞðÞ¼0: ð4Þ
By introducing a phasor
/ x; tðÞ¼/
Eq. 4 reduces to Laplace’s equation for the real and
imaginary parts of the phasor,
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/
þ j /
MATERIALS AND METHODS
Poly(ethylene glycol) diacrylate (PEG-DA, Mn ~
258), 2,2-Dimethoxy-2-phenylacetopheno ne (DMPA),
3-(Trimethoxysilyl)propyl acrylate (TPA), ethanol,
toluene, triethylamine, HEPES, calcium chloride,
D-glucose, sucrose, sodium hydroxide, saponin,
b-mercaptoethanol, and gelatin were purchased from
Sigma-Aldrich (St. Louis, MO). (Tridecaﬂuoro-
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 Tec hnologies (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
(Newton, MA). Kno ck-Out Dulbecco’s modiﬁed
Eagle’s medium (KO-DMEM), phosphate buffered saline
(PBS), ﬁbronectin, trypsin,
streptomycin, non-essential amino acids (NEAA), an d
LIVE/DEAD viability/cytotoxicity kit were purchased
FIGURE 1. Proposed cell patterning method using positive DEP force deﬁned by non-fouling microstructures. (a) Positive DEP by
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.
FIGURE 2. Finite element analysis of positive DEP force on a mammalian cell. (a, b) Computational domain (1000 lm 3 100 lm)
and boundary conditions for solving Laplace’s equation (Eq. 5). The microwell’s diameter and thickness are 100 lm and 10 lm,
5 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).
Efﬁcient Dielectrophoretic Patterning 3779
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: cu ring 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
was passivated with vapor-phase (tridecaﬂuoro-
PEG microwells were fabricated on an ITO glass
slide which served as the bottom plate of the micro-
ﬂuidic chann el as well as the bottom elect rode. First,
ITO surface was functionalized with acrylate groups
for covalent bonding with PEG-DA structures
(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 wer e 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. 3 b). 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/cm
of UV light for 60 s
using a UV spot cure system (EXFO OmniCure S1000,
EXFO, Ontario, Canada) (Fig. 3d). After removal of
the PD MS 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. 3 f). 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
FIGURE 3. Fabrication of a microﬂuidic channel with an
embedded PEG microwell array and integrated ITO
TSUTSUI et al.3780
each piece. Inlet and outlet holes were punch ed
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 LW1
were 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% CO
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,
D-glucose, and 236 mM sucrose,
and its pH
was adjusted to 7.35 by NaOH
, 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)
level (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 V
at 10 MHz) was applied between the electrodes by a
function generator (33120A, Hewlett-Packar d, 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% CO
) 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
cells remain unstained. Cell viability data was
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 cultur e
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 membr ane of non-viable cells and stain red after
binding to the nucleic acids. Fluorescence images of
the stained cells were captured using the inverted
ﬂuorescence microscope and the CCD camera
Efﬁcient Dielectrophoretic Patterning 3781
RESULTS AND DISCUSSIONS
Finite-Element Method Analysis
According to published experimental work,
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.
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.
List 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. Dis tribution of the DEP
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
FIGURE 4. Flow cytometry analysis of mES cell viability after a 30-min incubation in LCB. Routinely cultured mES cells were
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.
TABLE 1. Electrical properties of materials and cell
Dielectric constant e
= 8.854 9 10
LCB 0.020 80
Cell membrane 3 9 10
Cytoplasm 0.5 50
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 intr oduced to the microwell array secti on, the
ﬂow was stopped and DEP patterning was initiated by
applying an AC potential (20 V
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: 10
FIGURE 5. DEP manipulation of polystyrene beads (negative DEP) and mES cells (positive DEP) without an external ﬂow under
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.
Efﬁcient Dielectrophoretic Patterning 3783
cells/mL. At the lower cell seeding density,
majority of cells in the ﬁeld of view imme diately
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
FIGURE 6. DEP patterning of mES cells with an external ﬂow, demonstrating quick capture and assembly of the cells and
formation of a viable homogeneous cell array. (a) At a lower cell seeding density (10
cells/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 20V
at 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 (10
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.
TSUTSUI et al.3784
bonds were formed between ITO a nd PEG-DA as well
as between glass and PDMS. On glass or silicon sub-
strates, PEG-DA hydrogel is typically immob ilized
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.
While 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 trimet hoxysilane molecules with a vinyl
terminal, including 10-undecenyltrimethoxysilane
Si) and vinyltrimethoxysilane (C
along with co ntrols (glass and ITO with or without
plasma treatment) and found that only the substrates
treated with 10-undecen yltrimethoxysilane 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.
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
FIGURE 7. Frequency dependence of DEP force and patterning efﬁciency. (a) Signiﬁcant reduction of positive DEP forces on
mES cells was observed at frequencies below 10 MHz. Seeding density: 10
cells/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 (DV
) 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
, meaning non-uniform electric ﬁelds required for DEP manipulation are equally attenuated. Scale bars: 200 lm.
Efﬁcient Dielectrophoretic Patterning 3785
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 E
in Eq. 1.
Hydrogel Materials Selection
In this study, PEG-DA 258 (Mn ~ 258) was chosen
as the building material of the microwel ls. 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.,
microwells 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) an d
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-
leading to poor structural stability of the
microwells anchored on substrates.
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
In 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 polyacrylamide
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 micro wells, 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.
Positive DEP-based patterning in this platform
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. Ther efore, total
consumption of cells cou ld 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 10
and membrane thickness d
(=8.0 9 10
having shell permittivity e
core permittivity e
þ2 r d
The CM factor was then calculated from Eqs. 2
and 7 using MATL AB (MathWorks, Matick, MA).
Parameter values used were adapted from the published
and 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
by assuming a simple model system
shown in the inset of Fig. 7c and using a corresponding
analytical so lution
where r, e, and h are conductivity, permittivity, and
height, respectively, and subscripts a and b indicate
corresponding domains, medium and PEG-DA,
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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