MICROFLUIDIC CELL CO-CULTURE USING STANDING SURFACE
ACOUSTIC WAVE (SSAW)
Sixing Li,a,b Feng Guo,a Yuchao Chen,a Xiaoyun Ding,a Peng Li,a Craig E. Cameron,b,c and
Tony Jun Huang*a,b
a
Department of Engineering Science and Mechanics, The Pennsylvania State University, University
Park, PA, 16802, USA.
b
Molecular, Cellular and Integrative Biosciences (MCIBS) Program, The Huck Institutes of the Life
Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
c
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University
Park, PA 16802.
ABSTRACT
The in vitro study of heterotypic cell-cell interactions requires co-culturing different types of cells in a
highly controllable manner. In this work, we demonstrate a standing surface acoustic wave (SSAW)-based
microfluidic device that is capable of patterning different types of cells sequentially in the generated SSAW
field to form organized cell co-cultures. In our approach, non-invasive acoustic forces are utilized to
manipulate cells in the SSAW field. The advantages of our SSAW-based microfluidic cell co-culture platform
include contactless cell manipulation, simplicity, high biocompatibility, high resolution, and minimal
interference of the cellular microenvironment. The microfluidic platform demonstrated here can be a valuable
tool for studying cell-cell interactions and multicellular tissue reconstruction.
KEYWORDS: Standing surface acoustic wave, cell co-culture, cell-cell interactions
INTRODUCTION
Cells residing in their in vivo niches interact with their neighboring cells to maintain normal cell function.
Thus, co-cultures of different cell types are needed to study cell-cell interactions and to reconstitute
multicellular tissue structure. Traditionally, different types of cells are randomly cultured together, which
lacks the precision and control needed to reconstruct physiologically relevant in vivo multicellular
microenvironment. To address these unmet needs, a cell co-culture technique with high biocompatibility, high
resolution, and minimal interference of the cellular microenvironment has yet to be realized.
Previously our group has demonstrated cell patterning using standing surface acoustic wave (SSAW).1–2
Our SSAW technique utilizes non-invasive, contactless acoustic forces to achieve high-precision and hightunability cell manipulation. These characters make our SSAW technique an ideal choice for cell co-culture
reconstruction. In this paper, we report a microfludic cell co-culture platform based on our SSAW technique.
Our SSAW-based cell co-culture platform can pattern different types of cells onto different regions to form
organized cell co-cultures. It has the advantages of contactless cell manipulation, simplicity, high
biocompatibility, high cell-patterning resolution, and minimal interference of cellular microenvironment. We
expect that the cell co-culture platform demonstrated here can be a valuable tool for studying cell-cell
interactions, tissue engineering, and drug screening.
THEORY
An optical image of our SSAW-based microfluidic cell co-culture device is shown in Figure 1(a), which is
made by bonding a polydimethylsiloxane (PDMS) microchannel in between a pair of interdigital transducers
(IDTs) fabricated on a lithium niobate (LiNbO3) piezoelectric substrate.
The basic mechanism of our SSAW-based cell co-culture platform is shown in Figure 1(b). The pair of
IDTs are aligned in parallel. When a radio frequency (RF) signal is applied to both IDTs, two series of
identical surface acoustic waves (SAWs) propagate in opposite directions to form a SSAW field, with a
periodic distribution of pressure nodes (with minimum pressure amplitude) and pressure antinodes (with
maximum pressure amplitude) on the piezoelectric substrate. When the resonating SSAW encounters the
liquid medium inside the microchannel, it generates longitudinal-mode leakage waves and causes pressure
fluctuations inside the medium. As a result, cells suspended in the culture medium will be aligned in parallel
lines in the established SSAW field by the acoustic radiation forces when the SSAW is on [Figure 1(c)].
When the SSAW is off, cells eventually settle down inside the microchannel and, in the absence of external
flow, maintain their original pattern [Figure 1(d)]. Eventually, these cells will attach to the surface of the
978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001
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18th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 26-30, 2014, San Antonio, Texas, USA
piezoelectric substrate forming a patterned cell
culture. This is the basis for co-culturing different
types of cells in our SSAW-based microfluidic cell
co-culture device.
EXPERIMENTAL
HeLa cells and NIH 3T3 fibroblasts were
maintained in DMEM/F12 medium (Gibco),
supplemented with 10% fetal bovine serum (Gibco)
and 1% penicillin-streptomycin (Mediatech). A CO2
incubator (NuAire) was used to maintain a
temperature of 37°C and a 5% CO2 level during cell
culture. For microfluidic cell co-culture experiments,
HeLa cells and NIH 3T3 fibroblasts were labelled
with green (CellTracker® Green CMFDA) and red
fluorescence dyes (CellTracker® Orange CMRA),
and cultured together in our SSAW device.
All the experiments were conducted on the stage
of an inverted microscope (Nikon TE2000U)
installed with an Chamlide® microscope incubation
system (Live Cell Instrument, South Korea). A
function generator (AFG 3102C) and power amplifier
(Amplifier Research 25A250A) were used to apply
RF signals to form the SSAW. A syringe pump
(KDS210, KD Scientific) was connected to the
microchannel to infuse fresh culture medium during
long-term cell culture. An ORCA-Flash 2.8 camera
(Hamamatsu, Japan) connected to the microscope
was used for data acquisition. Image processing was
conducted using Image J (NIH, Bethesda, MD).
Figure 1: (a) An optical image of our SSAW-based
microfluidic cell co-culture device. (b) The basic
mechanism of our SSAW-based cell co-culture
platform. (c) Patterned HeLa cells in suspension
with the SSAW on. (d) Patterned HeLa cells
adhered to the bottom surface with the SSAW off.
RESULTS AND DISCUSSION
We first explored the possibility of forming
organized cell co-cultures with one round of cell
patterning. In this experiment, green fluorescently-labelled HeLa cells were first introduced into the
microchannel at a seeding density of 4 × 106 cells/ml. When a 12.78 MHz RF signal with a voltage of around
20 Vpp was applied to the IDTs, the HeLa cells were patterned in parallel lines with a period of ~150 μm.
After 3 h of culture, the HeLa cells formed a patterned cell growth, as shown in Figure 2(a). Then red
fluorescently-labelled NIH 3T3 fibroblasts were injected into the microchannel at the same seeding density.
Without the SSAW applied, NIH 3T3 fibroblasts adhered and grew with a random distribution after another 3
h of culture, as shown in Figure 2(b). As a result, a co-culture of HeLa cells and NIH 3T3 fibroblasts was
formed, in which HeLa cells were in an organized pattern while NIH 3T3 fibroblasts were in a random
distribution [Figure 2(c)].
Although cell co-cultures can be formed with one round of cell patterning, the positions of different types
of cells are not well-controlled. Therefore, we further examined the possibility of patterning and co-culturing
different types of cells. In the established SSAW field, the distribution of pressure nodes and antinodes can be
changed by tuning either the excitation frequency or the relative phase shift of the applied RF signals. In our
experiment, we chose the latter method so that different types of cells can be patterned and cultured in a
sequential manner.
Figure. 3 shows the experimental results validating this sequential cell patterning approach. In this
experiment, green and red fluorescently-labelled HeLa cells were used to represent two cell types. We first
introduced the green fluorescently-labelled HeLa cells into the microchannel at a seeding density of 4 × 106
cells/ml and patterned the cells under an RF signal of 12.78 MHz, 20 Vpp, and 0° phase shift. After 2 h of
culture with the SSAW off, we injected the red fluorescently-labelled HeLa cells into the microchannel at the
same seeding density. These cells were patterned under an RF signal of 12.78 MHz, 20 Vpp, but with a 180°
phase shift. After another 2 h of culture with the SSAW off, fluorescent images were taken. As shown in
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Figure 3: Cell co-culture with sequential cell patterning. (a)
Mechanism of patterning two types of cells in different positions
with phase shift approach. (b) Green fluorescent image showing
firstly-seeded HeLa cells. (c) Red fluorescent image showing
secondly-seeded HeLa cells. (d) Merged image showing green
and red HeLa cells grown in alternating lines.
Figure 2: Cell co-culture with one
round of cell patterning. (a) Green
fluorescent image showing patterned
HeLa cells. (b) Red fluorescent
image showing randomly-seeded
NIH 3T3 fibroblasts. (c) Merged
image showing the co-culture of
HeLa cells and NIH 3T3 fibroblasts.
Figure. 3(a), the change of the relative phase shift between the pair
of IDTs from 0° to 180° will change the cell-patterning positions
between the two rounds of cell seeding by switching between
pressure nodes and antinodes. The fluorescent images in Figure.
3(b-c) show the two groups of HeLa cells growing in patterned
lines. From the merged image in Figure. 3(d), we can see that a
well-organized co-culture of the two groups of HeLa cells was
formed.
CONCLUSION
In summary, we have developed a SSAW-based microfluidic cell co-culture platform. Our SSAW-based
microfluidic cell co-culture platform has the advantages of contactless cell manipulation, simplicity, high
biocompatibility, high resolution, and minimal interference of the cellular microenvironment. The
microfluidic cell co-culture platform demonstrated here has great application potential in cell communication
studies, tissue engineering, and pharmaceutical research.
REFERENCES
[1] J. Shi, D. Ahmed, X. Mao, S.-C. S. Lin, A. Lawit, and T. J. Huang, Acoustic tweezers: patterning cells
and microparticles using standing surface acoustic waves (SSAW), Lab Chip, Vol. 9, pp. 2890-2895,
2009.
[2] X. Ding, J. Shi, S.-C. S. Lin, S. Yazdi, B. Kiraly, and T. J. Huang, Tunable patterning of microparticles
and cells using standing surface acoustic waves, Lab Chip, Vol. 12, pp. 2491-2497, 2012.
CONTACT
*Tony Jun Huang, E-mail: [email protected]
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