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MEASURING THE IMPACT OF DIELECTROPHORESIS ON CELL PHYSIOLOGY USING A
HIGH-CONTENT SCREENING PLATFORM
Salil P. Desai and Joel Voldman
Massachusetts Institute of Technology, Cambridge, MA, USA
ABSTRACT
Here we show the design and implementation of a microfabricated platform for
screening the effects of electric fields on mammalian cells. Specifically, we have
screened for electric field conditions that are commonly used in the manipulation of
cells using dielectrophoresis. To assay the physiological state of the cells, we
constructed and cloned a stress-reporting cell line. Results obtained with this
platform indicate that both high field strengths and low-frequency fields adversely
affect cell physiology.
KEYWORDS: Dielectrophoresis, stress, physiology, high-content screening.
INTRODUCTION
Dielectrophoresis (DEP) is an
important
technique
for
the
manipulation of cells [1]. Although
researchers have studied the gross
effects of DEP on cell viability and
growth [2], little is known about how
electric fields couple through
intracellular signaling pathways to
alter cell physiology. It is well
known that electric fields used for
DEP can not only stress cells via
temperatures rises due to Joule
heating of the culture media, but can
also potentially directly interact with
cells (e.g., via voltage-gated ion
channels) [3]. Consequently, it is
imperative that we understand the
effects of DEP manipulation on cell
physiology to determine whether
DEP manipulation itself can alter
particular phenotypes of interest and
confound downstream biological
assays.
To this end, we have
developed a microfabricated, highcontent screening platform that can
apply a large number of different
electrical stimuli to cells and then
Figure 1: Device design and E-field
generation. (A) Schematic (not to scale)
of top-view of an array of 16 electrode
sites. (B) Technique for preparing device
for screen. (C) Images of electrode chips
(left), bottom electrode (inset) and
packaging setup (right). (D) A customdesigned computer-controlled relay box
switches sinusoidal waveforms from a
function generator to each individual
electrode site.
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
978-0-9798064-1-4/µTAS2008/$20©2008CBMS
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monitor the molecular effects of those stimuli using automated fluorescence
microscopy.
EXPERIMENTAL
The platform consists of a chip with
individually
addressable
arrayed
electrodes and support electronics to
generate the desired waveforms (Figure
1A, C). Mammalian cells are seeded on
the chip (Figure 1B) and then the entire
assembly is clamped and placed in a
standard cell culture incubator, where a
Figure 2. Stress reporter cell line.
computer-controlled
custom-designed
(A) Reporter construct designed
switch
box
automatically
and
using co-transfection of plasmids.
autonomously
applies
arbitrary
(B) Fluorescence images of cells
stimulation waveforms (varying voltage,
showing constitutive red expression
frequency, and duration) to individual
and inducible green expression.
electrode sites (Figure 1D).
Scale bar 25 μm.
The platform can use any fluorescent
reporter cell line to assess the impact of the applied fields. We specifically
engineered a reporter cell line that monitors the cells’ general stress response
pathway. This reporter
cell line expresses green
fluorescent
protein
(GFP) under the control
of a heat shock element
(HSE)
promoter
construct [4]. The HSE
promoter regulates the
stress response under
thermal, chemical, and
other
environmental
stresses. The reporter
shows a 10× increase in
GFP fluorescence in
response
to
stress,
Figure 3. Imaging and assay readout. Composite
providing a high signalimage (left) showing multiple images from a single
to-noise ratio for the
electrode. Via multi-wavelength fluorescence imaging
detection of stressed
of each electrode, we use DsRed images to mask the
states.
Further, the
GFP images and extract quantitative information
response
is
dosefrom single cells in the GFP image. Scale bar 40 μm.
dependent, allowing us
to quantify the amount of stress by the fluorescence intensity. These reporter cells
also constitutively express a red-fluorescent protein (DsRed), ensuring that cells
expressing low levels of GFP can still be identified and imaged. The robust
fluorescence signal of these stress-reporting cell lines allow for the use of automated
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
1309
microscopy techniques for acquiring data (Figure 3) and the use of image processing
algorithms to extract quantitative information from single cells.
RESULTS AND DISCUSSION
We have used this system to assess the stress response of cells to varying
stimulus voltages, frequencies, and durations (Figure 4). The voltage sweep shows
Figure 4. Effects of Electric fields. (A) The voltage sweep shows a dramatic
increase in cellular stress with increase in voltage. (B) A frequency sweep
indicating that cells are stressed at low frequencies (possibly due to
transmembrane loading effects). (C) A heat map showing a voltage sweep for
different durations of field exposure. Longer durations of exposure show
increased cellular stress levels.
an increase in stress with voltage, which is to be expected as increasing voltage will
increase heating and thus thermal stress. The frequency sweep shows a slight
decrease with increasing frequency, perhaps due to lower transmembrane potentials
at higher frequencies. Finally, the voltage-vs.-duration heat map shows that one can
trade off higher voltage for shorter duration to exact the same stress.
CONCLUSION
This platform is the first system that enables the quantification of the impact of a
wide range of electrical field strengths, frequencies and durations on cell physiology
at a molecular level. With this platform we can provide both maps of physiologically
optimal conditions for DEP manipulation and probe the fundamental biological
processes that are modulated by electrical fields.
REFERENCES
[1]
Voldman, J. Electrical forces for microscale cell manipulation. Annu. Rev.
Biomed. Engr., 8, 425- 454 (2006).
[2]
Fuhr, G., Glasser, H., Muller, T. & Schnelle, T. Cell manipulation and
cultivation under AC electric-field influence in highly conductive culture
media. Biochim. Et Biophy. Acta, 1201, 353-360 (1994).
[3]
Marszalek, P., Liu, D. S., and Tsong, T. Y. Schwan equation and
transmembrane potential induced by alternating electric fields, Biophys. J.,
58, 1053-1058 (1990).
[4]
Vasanawala, F., Tsang, T., Fellah, A., Yorgin, P., and Harris, D.T. A novel
expression vector induced by heat, γ-radiation and chemotherapy, Gene
Ther. Mol. Biol., 5, 1-8 (2000).
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October 12 - 16, 2008, San Diego, California, USA
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