SIMULTANEOUS DIELECTROPHORETIC TRAPPING OF CELL ON
OPPOSITE SIDES OF A PERMEABLE MEMBRANE
1
B.J. Nablo and D.R. Reyes*
National Institute of Standards and Technology, USA
ABSTRACT
Molecular transport through permeable membranes offers a unique opportunity to investigate cellular
responses to nutrients, drugs, or toxins delivered to the culture’s apical and/or basal sides as well as
cellular migration events. Herein, we present the means to rapidly localize and concentrate cells using
dielectrophoresis (DEP) traps on opposing sides of a permeable membrane within a multilayer
microfluidic device. This work establishes the possibility of generating on-chip cell co-cultures for highthroughput screening, similar to transwell chambers, but with the added benefits of the dynamic fluidic
conditions from the microfluidic network and the rapid generation of cell cultures from DEP trapping.
KEYWORDS: Multilayer microfluidic system, co-culture, dielectrophoresis, permeable membrane
INTRODUCTION
A standard macroscopic method to study molecular transport through permeable membranes involves
the transwell/Boyden chambers with static reservoirs. The permeable membranes provides a suitable
environment to study cellular migration, cell-cell communication, and cellular responses to nutrients,
drugs, or toxins delivered to the cell in a 3-dimensional fashion with independent solution control on both
sides of the membrane. A microfluidic device that includes a permeable membrane as a separator could
extend the capabilities of transwell chambers while providing the advantages of microfluidics (Fig.1).
Other research has shown that cells can be assembled into discrete cell layers with [1] or without [2] a
membrane as a separation layer. With the incorporation of electrode arrays, our device provides the
advantage of direct cell loading via DEP trapping in order to rapidly position and generate cell cultures.
Moreover, the DEP forces exerted on the cells can overcome gravity, allowing for the simultaneous
loading cells on the opposite sides of the membrane. We demonstrate the attachment of cells on opposite
sides of a permeable membrane within microfluidic devices, and the cells remained viable until they
obstruct the microchannel ( > 5 days).
EXPERIMENTAL
We have previously shown that DEP forces trap cells at patterned gold electrodes on a polyester
membrane located within a microchannel [3]. Improvements to standard photolithography technics
enhanced the integrity of the interdigitated 50-nm thick gold microelectrodes, so that two arrays could be
patterned and aligned on opposite sides of a porous PET membrane. The final product is a highly-flexible
electronic system that can withstand radius of curvatures of a few millimeters. In the assembled devices
(Fig. 1), the electrode arrays are suspended across the intersection of two perpendicular
polydimethylsiloxane (PDMS) microchannels, where the top electrodes become the floor of the top
channel and the bottom electrodes are the ceiling of the bottom channel. The device is bonded by
oxidizing the PDMS surfaces.
Prior to cell trapping, a hybrid cell adhesion material (hCAM) of fibronectin (Sigma-Aldrich) [4] and
polyallylamine hydrochloride (PAH, Sigma-Aldrich) coats the microchannels in order to promote
adhesion of the trapped cells to the membrane surface [5]. It is noteworthy that when the hCAM is
absent, cells can be trapped and released at will. Cells were prepared for trapping by harvesting the cells
from tissue culture flasks, centrifuging the suspension, decanting the supernatant, and re-suspending the
cells in an electrolyte-free, osmolality-similar sucrose solution, which amplifies the DEP trapping forces
exerted from a < 2 Vp-p, 10 MHz sine wave at the electrode arrays. Cell culture media was exchanged
within < 15 minutes after re-suspension. After trapping, the appropriate cell media was exchanged for the
respective channel. After 3-5 days, cell viability dyes Calcein AM (Life Technologies) and Dead Red
978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001
549
19th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 25-29, 2015, Gyeongju, KOREA
500
µm
Figure 1. The dual DEP device. (Left Panel) Optical image (rotated 90 degrees) of the two independent, concentric, interdigitated electrode arrays. The double-sided electrode array is suspended between the top (red arrow)
and bottom (green arrow) 40-µm high microfluidic channels. The circular array is closest to the objective (ceiling of the bottom channel), while the diamond is on the opposite side of the membrane (floor of the top channel).
The pores in the membrane are visible as the black speckles. (Right Panel) The assembled device consisting of
gold electrodes patterned on both sides of a permeable PET membrane placed between two PDMS microfluidic
networks (green v. yellow).
(Life Technology) were added to one or both microchannels in order to better visualize the cells and
determine cell viability.
RESULTS AND DISCUSSION
Periodic and simultaneous DEP forces successfully trapped for the co-localization of homo- and
hetero-cultures of hepatocytes (HepG2), fibroblast (NIH-3T3), and endothelial cells (Human Umbilical
Vein Endothelial Cells, HUVEC), regardless of osmolality differences of supporting solutions.
Exemplified in Figure 2, HepG2 cells and HUVECs are trapped simultaneously at the floor of top channel
(diamond electrode) and the ceiling of the bottom channel (circle electrode), respectively.
After replacing the sucrose with the appropriate media in both channels, cells proliferate and survive
for more than 5 days (Fig. 3). Despite trapping against gravity, the HepG2 cells at the ceiling of the
bottom channel remain adhered and proliferated normally along the ceiling when hCAM is present. Note,
the cell orientation is inverted between Figure 2 and Figure 3. After supplying fluorescent dye to only the
top microfluidic channel (Fig. 3), the cells in the bottom channel are able to uptake the dye only at and
downstream from the suspended membrane region, thus demonstrating that both cell lines cells are viable
and the membrane pores are unobstructed.
The porosity of the PET membrane grants the passage of sub-micron solution components at the
crossover region of the channel, allowing for the potential study of cell-cell communication or indirect
exposure of small molecules such as drug candidates, toxins and metabolites.
CONCLUSION
This work demonstrates the use of photolithography to pattern gold electrodes on both sides of
permeable PET membranes to produce a highly-flexible electronic system capable of DEP trapping on
both sides of PET membranes. Using DEP and hCAM, cells were trapped simultaneously on opposite
sides of the PET membrane and held in place in the presence of a fluid flow field even after abating the
electric field. This work demonstrates the application of DEP trapping to immobilize cells on both sides
of a porous membrane to produce a co-culture system in a microfluidic device. The potential uses of this
platform span from the study of cell co-cultures, such as cell-cell communication and cell migration,
where conditions can be independently optimize to provide the best growth and function conditions for
each cell line.
550
500 µm
500 µm
Figure 3. Co-localized cultures of two cell
lines 84 h after DEP trapping and live/dead staining via only the top channel only. HUVECs were
trapped at the diamond electrode located on the
floor of the top channel, while HepG2 hepatocytes were trapped at the circle electrode located
on the ceiling of the bottom channel. Media was
exchanged every 12 h. After 84 h, a live/dead cell
viability dyes were introduced to the HUVEC media only (green arrow), while the HepG2 media
remained dye-free (red arrow). The Dead Red dye
stains the PET membrane of the entire top channel, while the Calcein-AM stains all the viable
HUVECs (diffuse green ovals). The dyes permeate into the bottom channel at the suspended region to stain the HepG2 cells (dense green circles), while the PET downstream is lightly stained
by the Dead Red.
Figure 2. Co-localization of two cell lines using
the dual-sided DEP microfluidic device. (Top)
HepG2 hepatocytes (red) are trapped at the diamond electrode located on the floor of the top
channel, while HUVECs (green) are trapped at
the circle electrode located on the ceiling of the
bottom channel. (Bottom) A cartoon of the device cross-section in operation (not to scale).
The membrane is shown in grey with the gold
electrodes patterned on both sides. The arrows
denote the direction of fluid flow in the bottom
(left to right, red) and the top (into the page,
green) channels.
ACKNOWLEDGEMENTS
This research was performed in part at the NIST Center for Nanoscale Science and Technology.
REFERENCES
[1] D. Huh, et al. “Reconstituting Organ-Level Lung Functions on a Chip,” Science, 328, 1662-1668,
2010.
[2] W. Tan and T.A. Desai, “MIcroscale multilayer cocultures for biomimetic blood vessels,” J. Biomed. Mater. Res., 72A, 146–160. 2005.
[3] C. Hanke, P.S. Dittrich, and D.R. Reyes, “ Dielectrophoretic Cell Capture on Polyester Membranes,” ACS
Appl. Mater. Interfaces, 4, 1878-1882, 2012.
[4] Certain commercial equipment, instruments, or materials are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment is necessarily the
best available for the purpose.
[5] D.R. Reyes, J.S. Hong, J.T. Elliott, and M. Gaitan, “Hybrid Cell Adhesive Material for Instant Dielectrophoretic Cell Trapping and Long-Term Cell Function Assessment,” Langmuir, 27, 10027-10034, 2011.
CONTACT
* Dr. Darwin R. Reyes; phone: +1-301-975-5466; darwin.reyes@nist.gov
551