TITLE:
Designing microfluidic devices for
studying cellular responses under single or coexisting chemical/electrical/shear
stress stimuli
AUTHORS:
Chou, Tzu-Yuan
Department of Agricultural
Chemistry
National Taiwan University
Taipei, 10617, Taiwan
b99603003@ntu.edu.tw
Sun, Yung-Shin
Department of Physics
Fu-Jen Catholic University
New Taipei City 24205, Taiwan
Hou, Hsien-San
Research Center for Applied Sciences
Academia Sinica
Taipei City 11529, Taiwan
Wu, Shang-Ying
Department of Agricultural
Chemistry
National Taiwan University
Taipei, 10617, Taiwan
Zhu,
Yun
Department of Agricultural
Chemistry
National Taiwan University
Taipei, 10617, Taiwan
Cheng, Ji-Yen
Research Center for Applied Sciences
Academia Sinica
Taipei City 11529, Taiwan
Lo, Kai-Yin
Department of Agricultural
Chemistry
National Taiwan University
Taipei, 10617, Taiwan
CORRESPONDING AUTHOR:
Lo, Kai-Yin
KEYWORDS:
microfluidic chip,
cell
migration, reactive oxygen species, electric field, electrotaxis, shear stress
SHORT ABSTRACT:
Micro-fabricated
devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based
microfluidic chips for studying cellular responses under single or coexisting
chemical/electrical/shear stress stimuli.
LONG ABSTRACT:
Microfluidic devices are capable of creating a precise
and controllable cellular micro-environment of pH,
temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in
vitro cell studies by providing in
vivo-like surroundings. Especially, how cells response to chemical
gradients, electrical fields, and shear stresses has drawn many interests since
these phenomena are important in understanding cellular properties and
functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers,
polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET)
substrates. Out of these
materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic
devices have been designed and fabricated for generating multiple, coexisting
chemical and electrical stimuli, none of them was considered efficient enough
in reducing experimental repeats, particular for screening purposes. In this report,
we describe our design and fabrication of two PMMA-based microfluidic chips for
investigating cellular responses, in the
production of reactive oxygen species and the migration, under single
or coexisting chemical/electrical/shear stress stimuli. The first chip
generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture
regions, together with a shear stress
gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each
culture area. These devices not only provide cells with a precise, controllable
micro-environment but also greatly increase the experimental throughput.
INTRODUCTION:
In vivo cells are surrounded by a variety of biomolecules including
extracellular matrix (ECM), carbohydrates, lipids, and other cells. They
functionalize by responding to micro-environmental stimuli such as interactions
with ECM and responses to chemical gradients
of various growth factors. Traditionally, in vitro cell studies are conducted in cell culture dishes where
the consumption of cells and reagents is large and cells grow in a static
(non-circulating) environment. Recently, micro-fabricated devices integrated
with fluidic components have provided an alternative
platform for cell studies in a more controllable way. Such devices are capable
of creating a precise micro-environment of chemical and physical stimuli while
minimizing the consumption of cells and reagents. These microfluidic chips can
be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS)
polymers, polymethylmethacrylate (PMMA) substrates, or
polyethyleneterephthalate (PET) substrates 1-3. PDMS-based devices are transparent, biocompatible, and
permeable to gases, making them suitable for long-term cell
culture and studies. PMMA and PET substrates are cheap and easy to be processed
using laser ablation and writing.
Microfluidic devices should provide cells with a stable and
controllable micro-environment where cells are subject to different chemical
and physical stimuli. For example, microfluidic chips are used to study
chemotaxis of cells. Instead of traditional methods that employ Boyden chamber and capillary 4,5 these miniaturized fluidic devices can generate precise
chemical gradients for studying cells’ behaviors1,6,7.
Another example is to study cells’ directional migration under electric fields
(EFs), a phenomenon named electrotaxis. Electrotactic behaviors of cells
were reported to be related to nerve regeneration8, embryonic development 9, and wound healing 10,11. And many studies have been performed to investigate the electrotaxis of various cell types including cancer cells 12,13, lymphocytes14,15, leukemia cells 11, and stem cells 16. Conventionally, Petri
dishes and cover glasses are used to construct electrotactic chambers for
generating EFs 17. Such simple setups pose problems of medium evaporation and imprecise
EFs, but they can be overcome by
microfluidic devices of enclosed, well-defined fluidic channels12,18,19.
To systematically study cellular responses under precise, controllable chemical and electrical stimuli, it would be of great use to develop
microfluidic devices capable of providing cells with multiple stimuli at the same time. For
example, Li et al. reported a PDMS-based microfluidic device for creating single or coexisting chemical gradients and EFs20. Kao et al. developed a
similar microfluidic chip to modulate the chemotaxis of lung
cancer cells by EFs6. Moreover, to increase the throughput, Hou et al. designed and fabricated a PMMA-based multichannel-dual-electric-field chip to provide cells with 8 different
combined stimuli, being (2 EF
strengths × 4 chemical concentrations)21. To further increase the throughout and add the
shear stress stimulus, we developed two PMMA-based microfluidic devices for
studying cellular responses under single or coexisting
chemical/electrical/shear stress stimuli.
Reported by Lo et al.22,23, these devices contain five independent cell culture channels subject
to continuous fluidic flowing, mimicking the in vivo
circulatory system. In the first chip (the chemical-shear
stress chip or the CSS chip), five relative concentrations of 0,
1/8, 1/2, 7/8, and 1 are generated in the culture regions, and a shear stress
gradient is produced inside each of the five culture areas. In the second chip (the chemical-electric field chip or the CEF chip), by using one single set of electrodes and 2 syringe pumps, five EF
strengths are generated in addition to five different chemical concentrations
within these culture areas. Numerical calculations and simulations are
performed to better design and operate these chips, and lung cancer cells cultured inside these devices are
subject to single or coexisting stimuli for observing their responses with respect to the production of reactive
oxygen species (ROS), the migration rate, and the
migration direction. These
chips are demonstrated to be time-saving, high-throughput and reliable devices
for investigating how cells respond to various micro-environmental stimuli.
PROTOCOL:
1.
Chip design and
fabrication
1.1)
Draw patterns to be ablated on PMMA substrates and double-side tapes using commercial
software24.
1.1.1) To study the effects of
chemical concentrations and shear stresses, draw a “Christmas tree” pattern
with a varying width at its end in each of the five culture areas (Figure 1A
and 1B).
1.1.2) To study the effects of
chemical concentrations and electric fields, draw a “Christmas tree” pattern
with two more fluidic channels for salt bridges (Figure 2A and 2B).
1.2)
Scribe an individual pattern on a PMMA sheet or a double-side tape by
loading the corresponding file into the CO2 laser
scriber.
1.2.1) Turn on the laser scriber, and check its connection to the
computer. Open the pattern to be ablated using the commercial software.
1.2.2) Place a PMMA sheet or a double-sided tape on top of the stage of
the scriber. Adjust the focus of the CO2 laser if necessary using the
calibration bar and the visible He-Ne laser.
1.2.3) Load the pattern into the scriber for ablation on the PMMA sheet
or the tape.
1.2.4) Pick up the patterned sheet or tape, remove unwanted pieces, and
clean the surface with nitrogen blowing.
NOTE: The thickness of the PMMA
sheet is 1 mm, and that of the double-sided tape is 0.07 mm or 0.22 mm.
2.
Chip assembly
2.1)
With super glue, attach acrylic adaptors (length × width × height = 10
mm × 10 mm × 5 mm, with screw threads in
the middle) to the top-most layer of the chip by aligning the screw threads and
the holes on the top-most layer. These adaptors serve as medium inlets/outlets
and salt bridge connectors.
2.2)
Assemble the microfluidic chip inside a laminar flow hood.
2.2.1) To assemble the chemical-shear stress chip (CSS chip), attach
three PMMA sheets together via two double-sided tapes of thickness = 0.07 mm
(Figure 1A and 1B).
2.2.2) To assemble the chemical-electric field chip (CEF chip), attach
the PMMA sheet to a double-sided tape of thickness = 0.22 mm (Figure 2A and
2B).
2.3)
Attach the chip onto a 10-cm cell culture Petri dish.
2.3.1) Attach the CSS chip to the Petri dish via one more double-sided
tape of thickness = 0.07 mm.
2.3.2) Attach the CEF chip to the Petri dish via the original 0.22 mm
double-sided tape.
2.4)
Leave the assembled chip inside the hood and expose it to UV for 30 minutes
for sterilization. Connect the inlets to two 3-ml syringes via plastic tubes and finger-tight nuts. Connect
the outlet to a waste tube via a plastic tube and a finger-tight nut.
2.5)
Prepare agar salt bridges for
generating electric fields in the CEF chip.
2.5.1) Autoclave all tubes and nuts at 121 °C for 15
min prior to usage. Dissolve 3% low melting point agarose in 1× phosphate
buffered saline (PBS) buffer using a microwave. Inject
solution-phased agarose into the salt bridge channel and insert the electrodes
before solution solidifies.
2.6)
Connect the syringes to the syringe pumps and continuously flow 1× PBS into the fluidic channel for
1 or 2 min at a flow rate of 20 μl/min. Disconnect the syringes from the
pumps and put the chip inside an incubator overnight
under 5% CO2 at 37 °C.
NOTE: These two steps are aimed to wash the chip and remove any remaining
bubbles within the chip.
2.7)
Take the chip out of the incubator, connect the syringes to the syringe pumps,
and continuously flow the culture medium (Dulbecco’s Modified Eagle’s medium, DMEM) into the fluidic channel for 1 or 2 min at a flow
rate of 20 μl/min to replace PBS.
3.
Cell preparation and
experimental setup
NOTE: Pre-warm 1× PBS, culture medium (DMEM plus
FBS), and trypsin in a 37 °C water bath
before usage.
3.1)
Plate 2 × 105 lung cancer CL1-5 cells25,26 in a 10-cm Petri dish supplied with
DMEM plus 10% fetal bovine serum (FBS). Incubate the cells inside an incubator under 5% CO2
at 37 °C until 90% confluence.
3.2)
Aspirate the medium and wash the cells once with
pre-warmed 1× PBS. Add 2 ml of 10% trypsin buffer to the cells and wait for 2 to 3 minutes
at 37 °C to detach
the cells.
3.3)
Transfer the cells to a 15-ml sterile
centrifugation tube and add 6 ml of culture medium into the tube. Gently invert
the tube for mixing and take out 5 μl of the cell-containing medium for
counting the cell number in a hemocytometer.
3.4)
Centrifuge the tube at 300 ×g for 5 min. Suspend 106 cells in 1
3.5)
Infuse the cell-containing medium into
the microfluidic chip from the outlet and make sure that the solution
distributes all through the five culture areas. Disconnect the syringes from the pumps and incubate the chip inside an incubator under 5% CO2
at 37 °C for 2 hr.
4.
Experimental setup
4.1)
Take the chip out of the incubator and place it on top of a transparent indium tin oxide (ITO) glass heater.
NOTE: The ITO glass is connected to
a proportional-integral-derivative
(PID) controller for maintaining the temperature at 37 ± 0.5 °C via feedback from
a thermal coupler clamped tightly between the ITO heater and the chip.
4.2)
Put the chip-heater assembly on top of a motorized XY stage of an
inverted microscope for tracking cell migration or a fixed XY stage of an
inverted fluorescent microscope for measuring the production of ROS.
4.3)
To generate different chemical concentrations, fill
two syringes with 5 ml of chemical solutions of relative concentrations 0 and 1
(dissolved in culture medium) and pump them into the chip at desired flow rates: in the CSS chip, at a flow rate of 0.3 ml/min
for 1 hr; in the CEF chip, at a
flow rate of 20 μl/min for the first 20
4.4)
In the CEF chip, set up the electric fields.
4.4.1)
Place two agar salt bridges (the plastic tubes containing solidified
agarose) in two 50-ml beakers filled with 1× PBS. Place Ag/AgCl electrodes into the beakers. Connect the electrodes
to a power supply set at the
constant-current model in series with an ammeter.
4.5)
In the CEF chip, for tracking cell
migration, program the XY stage of the microscope to repeat taking photos, via
a digital single-lens reflex (DSLR)
camera, of certain field of views (FOVs) within culture areas every 15 min for
2 hr.
4.6)
For measuring the production of
ROS, use the fluorescence-based indicator 2′-7′-dichlorodihydrofluoresce
diacetate (2′-7′-DCFDA).
4.6.1) Prepare
the stock of 2′-7′-DCFDA at 10 mM in molecular
biology grade dimethyl
sulfoxide (DMSO).
Dilute DCFDA in DMEM only without serum (5
μM in DMEM). The potential deacetylase could increase the background
signals and decrease the signals in cells.
4.6.2) After 1 hr
of shear stress stimulus in the CSS chip or after 2 hr of EF stimulus in the
CEF chip, pump 2′-7′-DCFDA (5 μM in DMEM) into the chip at a
flow rate of 20 μl/min for the first 20
4.6.3) Take
photos, via a charge-coupled device (CCD) camera, of certain FOVs within culture areas for analyzing the fluorescent
intensities.
5.
Calculations of chemical concentrations, shear stresses, and electric
fields
5.1)
In both the CSS chip and the CEF
chip, calculate the chemical concentrations in the five culture areas. For
example, by injecting H2O2 of concentrations 0 and 200 μM
from the two inlets, concentrations of 0, 25, 100, 175, and 200 μM are
generated.
NOTE: By assuming
that all liquids split-flow smoothly and equally around the fork, the relative concentrations in the five culture areas are 0, 1/8, 1/2,
7/8, and 1, respectively.
5.2)
In the CSS chip, calculate the
shear stress (t) within each of the culture areas using
NOTE: By setting Q = 0.3 ml/min in each
inlet (Q = 0.12 ml/min
in each culture area), h = 0.0008 Pa×s for culture medium, h = 1 mm, and w = 1 ~ 4 mm, the
shear stress is calculated to range from 0.0048 Pa
(4 mm-wide region) to 0.0192 Pa (1
mm-wide region).
5.3)
In the CEF chip, calculate the EF
strength within each of the culture areas using E = I/(σAeff) (Ohm’s law), where I is the electric
current flowing across the fluidic channel, σ is the electrical
conductivity of the culture medium, and Aeff
is the effective cross-sectional area of the channel.
NOTE: Using σ = 1.38 Ω-1m-1 for culture medium
and Aeff = 0.22 mm2 (width =
1 mm and height = 0.22 mm), the EF strength
is calculated to be E (mV/mm)
= I (A) × 3.3 × 106.
5.3.1) As shown in Fig. 2D, treat the equivalent circuit as five C-section circuits with four (8
+ 35 + 8) segments and one (5 + 35 + 5) segment.
NOTE: By analyzing this parallel circuit according to Kirchhoff's
voltage law and Ohm's law, currents flowing across five
culture areas, I1 through I5 from
bottom to top, are calculated to be around
0.49I (area 1), 0.25I (area 2), 0.13I (area 3), 0.08I (area
4), and 0.05I (area 5), respectively, where I is the
total direct current (dc). With an
applied dc of 0.157 mA, EFs of 254, 130, 67, 41, and 26 mV/mm are
generated within the five culture areas.
NOTE: For a simplified electrical analysis of the microfluidic
network, all fluidic segments are
considered as resistors with resistance proportional to their
lengths.
6.
Data analysis
Note: Data analysis is performed using the ImageJ
software.
6.1) Analyze the production of ROS.
6.1.1) Run the
ImageJ software. Go to “File” à Open to load a fluorescent image to be analyzed.
6.1.2) Go to
“Image” à “Type” à “16-bit” to change the image
to a gray scale.
6.1.3) Draw
a polygon to enclose a cell. Go to “Analyze” à “Measure” to measure the mean fluorescent intensity of the cell.
6.1.4)
Repeat 6.1.3) to collect intensities from at least 50 cells of three
independent experiments, and calculate the mean intensity with standard error
of mean (SEM).
6.1.5)
Repeat 6.1.1) ~ 6.1.4) for each experimental condition.
6.2)
Analyze cell migration.
6.2.1) Run the ImageJ software. Go to “File” à Open to load an image taken at
time = 0 to be analyzed.
6.2.2) Draw a polygon to enclose a cell. Go to “Analyze” à “Measure” to measure the center
of mass of the cell as (x1,
y1).
6.2.3) Repeat 6.2.1) ~ 6.2.2) to measure the center of mass of the same
cell as (x2, y2) from another image take
at time = t.
6.2.4) Calculate the migration rate (in μm/hr) of this cell as
6.2.5) Repeat 6.2.1) ~ 6.2.4) to collect migration rates from at least
50 cells of three independent experiments, and calculate the mean migration
rate with standard error of mean (SEM).
6.2.6) From 6.2.2) and 6.2.3), calculate the migration
directedness of this cell as cosine θ or
6.2.7) Repeat 6.2.6) to collect migration
directedness from at least 50 cells of three independent experiments, and
calculate the mean migration directedness with standard error of mean (SEM).
6.2.8) Calculate the mean migration rate with SEM
and the mean migration directedness with SEM for each
experimental condition.
NOTE: A directedness of +1 indicates
that all cells migrate toward the cathode, and a -1 value indicates that all cells migrate toward the anode.
The directedness of a group of randomly moving cells is
close to 0.
6.3)
Analyze cell alignment.
6.3.1) Run the ImageJ software. Go to “File” à “Open” to load an image to be
analyzed.
6.3.2) Treat the cell as an ellipse and draw a line
to indicate the long axis of a cell. Go to “Analyze” à “Measure” to measure the angle β between the line and the
horizontal EF direction.
6.3.3) Repeat 6.3.2) to collect β from at least 50 cells of three
independent experiments, and calculate mean cosine β with standard error of mean (SEM).
6.3.4) Repeat 6.3.1) ~ 6.3.3) for each experimental
condition.
NOTE: A cosβ of +1 indicates
that all cells align in parallel to the applied
EF, and
a 0 value indicates
that all cells align perpendicularly to the
applied EF.
REPRESENTATIVE
RESULTS:
The chemical-shear stress (CSS)
chip
The CSS chip is made of
three PMMA sheets, each of thickness 1 mm, attached together via two
double-sided tapes, each of thickness 0.07 mm (Figure 1A and 1B). The “Christmas
tree” structure generates five relative concentrations of 0, 1/8, 1/2, 7/8, and
1 in the five culture areas. By designing the culture area as a triangle, a
shear stress gradient, with a magnitude related the volume flow rate, the
fluidic viscosity, and the dimension of the fluidic channel, is created within each
of the areas. This chip is then attached, via another 0.07 mm-thick
double-sided tape, to a Petri dish for culturing lung
cancer CL1-5 cells and the production of ROS was observed in response to
different chemical concentrations and shear stresses. The production of ROS in
lung cancer cells is highly related to lung cancer metastasis and development,
and certain chemicals and shear stress were demonstrated to be involved in ROS
generation 23.
First, to study the
effect of H2O2, a chemical stimulus, on the production of
ROS, cells were incubated with continuous flowing of H2O2
solutions at 0, 25, 100, 175, and 200 μM. As shown in Figure 1C, the fluorescent
intensity increased as the concentration of H2O2
increased, indicating that H2O2 stimulated the production
of ROS. Next, to investigate the effect of shear stress on the production of
ROS, cells were exposed to a shear stress gradient of 0.0048 Pa to 0.0192 Pa. Figure
1D shows that the fluorescent intensity increased as the shear stress increased
(the shear stresses were the highest and the lowest in the Front and Back
areas, respectively), suggesting that higher shear stress induced more ROS production. Also, this CSS chip was used to study the production of ROS in response to different concentrations
of α-tocopherol,
an antioxidant
of a form of vitamin E. Cells were stimulated by shear stress plus α-tocopherol of 0, 3.2, 12.5, 21.9, and 25
μg/ml. As shown in Figure 1E, for α-tocopherol concentrations lower than 21.9 μg/ml,
the fluorescent intensity
decreased as the concentration increased, indicating the effect of α-tocopherol
in reducing the production of ROS. However, as the concentration increased to 25 μg/ml, the mean intensity
also increased,
suggesting that this high
concentration of α-tocopherol did
not eliminate
much ROS
compared to lower concentrations.
The chemical-electric field (CEF)
chip
The CEF chip is made of a 1 mm-thick PMMA and a
0.22-mm double-sided tape with fluidic channels patterned on it (Figure 2A and
2B). Similarly, the “Christmas tree” structure
creates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the five
culture areas. In addition, these five parallel areas are connected
perpendicularly to form a fluidic path analogous to an electric circuit. The
electric field generated within a culture area is related to the conductivity
of the fluid, the cross-sectional area of the channel,
and the direct current (dc) passing through the area. According to
Kirchhoff's voltage law and Ohm's law, the
currents in the five culture areas are 0.49I, 0.25I,
0.13I, 0.08I, and 0.05I, respectively, where
I is the applied dc (Figure 2C). This chip is attached, via the 0.22 mm-thick
double-sided tape, to a Petri dish for culturing lung
cancer CL1-5 cells to observe cell migration and the production of ROS in
response to different chemical concentrations and electric fields.
First, this chip was used to study the production
of ROS in response to different concentrations of honokiol, different strengths of EFs, and
combined treatments of both. Honokiol, a small-molecule
polyphenol isolated from the genus Magnolia, was found to have
antiangiogenic, anti-inflammatory, and antitumor properties in preclinical studies 28. As shown in Figure 2D, the ROS produced was almost the same for EFs
lower than 67 mV/mm, but was increased with increasing EFs above this value.
Figure 2E shows that under combined treatments of electric fields and honokiol,
the ROS level stayed almost the same, indicating that honokiol inhibited
the production of exogenous ROS (i.e., ROS related to EF stimulus), especially
under higher EFs. The cell migration under single or
coexisting chemical/electrical stimuli was also investigated using this CEF
chip. As seen in Figure 2F, without the addition of honokiol, the migration rate increased
as the EF strength increased. After adding honokiol
of different concentrations, the migration rate decreased in general, suggesting
that honokiol reduced cell migration possibly via inhibiting the production of
ROS. The migration directedness is shown in Figure 2G. In the presence of
EF only, lung cancer cells showed prominent directional migration toward the anode (with negative
values). After adding honokiol of different concentrations, there was a slight decrease in migration directedness for all EF-stimulated areas.
Figure Legends:
Figure 1. The chemical-shear stress (CSS)
chip used to study the effects of chemicals and shear stresses on lung cancer cells.
23
(A) Three layers
of PMMA substrates are
bound together via two double-sided
tapes to form the CSS chip. (B) The integrated
CSS chip. (C) Top: Fluorescent and bright-field images of CL 1-5 cells after being treated with different
concentrations of H2O2. Scale bar
= 50 μm. Bottom: Mean fluorescent intensity with SEM plotted at different
concentrations of H2O2. (D) Top: Fluorescent
images of CL 1-5 cells under different shear stresses. Scale bar = 50 μm. Bottom: Mean fluorescent intensity with SEM plotted at
different shear stresses. (E) Top: Fluorescent and bright-field images of CL 1-5 cells after being stimulated with shear stress with different concentrations of α-tocopherol. Scale bar = 50 μm. Bottom: Mean fluorescent intensity with SEM plotted at
different concentrations of α-tocopherol. Data presented here was originally published in reference 23.
Figure 2. The chemical-electric field (CEF) chip used to study the
effects of chemicals and electric fields on lung cancer cells. 22
(A) One layer of PMMA is bound onto a culture dish via a double-sided
tape to form the CEF chip. (B) The fluidic pattern of the CSS chip. (C) The equivalent
electrical circuit of the microfluidic chip. (D) Left:
Fluorescent
and bright-field images of CL 1-5 cells after being treated with different strengths of EFs.
Scale bar = 50 μm. Right: Mean fluorescent intensity with SEM plotted at different
strengths of EFs. (E) Left: Fluorescent and bright-field images of CL 1-5 cells after being treated with
combined honokiol and EFs. Scale bar
= 50 μm. Right: Mean fluorescent intensity with SEM plotted at combined
honokiol and EFs. (F) The migration
rates of CL1-5 cells after being treated with EFs only (marked
as control) and combined honokiol and EFs (marked
as honokiol). (G) The migration directedness of CL1-5 cells after being treated
with EFs only (marked as control) and combined honokiol
and EFs (marked as honokiol). Data
presented here was originally published in reference 22.
DISCUSSION:
PMMA-based chips are fabricated using laser
ablation and writing which are cheaper and easier methods when compared to
PDMS-based chips which require more complicated soft lithography. After
designing a microfluidic chip, the fabrication and assembly can be done within
just 5 minutes. There are some critical steps that attention should be paid to
in performing the experiment. The first is the “assembling” issue. The adaptors
should be glued properly to the top-most layer of the chip. Glue could leak
into the fluidic channels if too much is applied, and liquid could leak out of
the chip if too little glue is used. Also, in assembling the PMMA substrates
and the double-sided tapes, it is important to apply pressure to press the
whole chip tightly to prevent any liquid leakage. The second is the “bubble”
issue. To remove bubbles, 1× PBS is continuously flowed into
the fluidic channel for 1 or 2 min at a flow rate of 20 μl/min and then
the chip is placed inside an incubator overnight under 5% CO2 at 37 °C. If there are bubbles remaining within the fluidic channels, the flow
will be non-uniform, causing nonhomogeneous chemical concentration and EF
distribution. The third is the “cell loading” issue. The number of cells loaded
into the culture areas should be well controlled. If the cell number is too
low, many experimental runs should be conducted to collect enough data for
statistical purpose. If the cell number is too high, it will be difficult to
distinguish individual cells and quantify their migration.
In the CSS chip, a “Christmas tree” structure
combined with a triangular culture area generates different chemical
concentrations in five areas, each having a shear stress gradient. Fluidic shear stresses were known to affect the attachment, the
morphology, the migration, and the activity of cells. Shear stresses of as low
as 0.25 ~ 0.6 Pa could interface cell attachment, and even higher values of
stresses (0.5 ~ 10 Pa) were reported to remove adherent cells 29. Laminar shear stresses ranging from 0.8 to 1.5 Pa induced cell
alignment in the direction of flow 30, and even lower
values (0.1 ~ 1 Pa) were known to affect cellular morphology and permeability 29. Moreover, smooth muscle cells subjected
to shear stresses of 2 ~ 120 Pa experienced a decline in cell number 31. Lu et al. reported the design and construction of microfluidic shear
devices for quantitative analysis
of cell adhesion 27. The shear stress within the fluidic channel was modified by changing
the dimensions of the channel. Chin et al.
described a hemodynamic Lab-on-a-chip system for controlling the flow rate of
the culture medium in the micro-channel to mimic the flow profile of the blood
in the vessel 32. The shear stress within the fluidic channel was modified by changing
the flow rate of the medium. In these two devices, even though a shear stress
of any values could be generated, only one single value was available within
one culture area at one time. By comparison, the present CSS chip has the
advantage of providing a shear stress gradient in one triangular area, increasing
the experimental throughput especially for a screening purpose.
In the CEF chip, the culture areas of the “Christmas
tree” structure are perpendicularly connected to form a fluidic path analogous to an electric circuit. At one applied direct
current, five EF strengths are generated inside the culture areas with each
having a flow of a specific chemical concentration. Conventionally, electrotactic experiments were performed on Petri dishes
where problems of imprecise EFs, medium evaporation, large cell/reagent consumption, and increased Joule heating could occur.
Enclosed, miniature microfluidic devices efficiently overcome these drawbacks.
For example, to study the electrotaxis of human blood memory T cells,
Lin et al. reported a plastic
microfluidic device containing two identical, side-by-side
micro-channels, two modified
pipette tips served as medium reservoirs,
and two platinum electrodes for EF application 15. To
increase the throughput, PMMA-based electrotactic chips were fabricated to provide multiple EF strengths in one single device12,33. Also, microfluidic
devices capable of generating controllable
chemical and electrical stimuli simultaneously are of high
interest. Li
et al. fabricated a PDMS-based
microfluidic device to generate
single or coexisting chemical gradients/EFs for investigating the chemotactic or electrotactic migration
of T cells14. Kao et al. employed
a similar microfluidic device to modulate the chemotaxis of lung
cancer cells by using EFs12. In these two devices, a Y-shape structure was applied to create a
stable concentration gradient in one single applied EF. Hou et al.
reported a multichannel-dual-electric-field chip to study
the concurrent effect of chemicals and EFs on lung cancer cells21. This device was able to provide 8 combinations of electrical/chemical stimuli
(2 electrical ×
4 chemical) in one experiment. Although the throughput
increased, this chip’s capacity was limited to (1) only one EF strength (plus
one zero as a control), and (2) four syringe pumps are required for pumping chemicals into four independent channels. By comparison, the present CEF
chip has the advantage of generating five EF strengths together
with five chemical concentrations via one single set of
electrodes and 2 syringe pumps.
Even though these chips are easy to be fabricated,
they have some limitations. First, only five “specific” concentrations and five
“specific” EF strengths can be generated. Second, the width of the fluidic
channel cannot be less than 1 mm due to the focusing of the CO2
laser beam. However, by precisely controlling the chemical concentration in the
inlet and the electric current passing through the fluidic channel, any
concentrations and EF strengths can be attained in the culture areas. In
conclusion, the present CSS and CEF chips are capable of providing cells with
controllable single or coexisting chemical/electrical/shear stress stimuli. In one
single CSS chip, five different chemical concentrations in combination with a
shear stress gradient can be generated. In addition, in one single CEF chip,
five different chemical concentrations in combination with five EF strengths
can be created. By increasing the branches of the “Christmas tree” structure,
the throughput of these chips can be further increased. These chips are
demonstrated to be an easy, time-saving, reliable, and high-throughput platform
for studies of cellular behavior under chemical/electrical/shear stress
stimuli.
ACKNOWLEDGMENTS:
This
work was financially supported by the Ministry
of Science and Technology of Taiwan under Contract No. MOST 104-2311-B-002-026
(K. Y. Lo), No. MOST 104-2112-M-030-002
(Y. S. Sun), and National Taiwan University Career Development Project
(103R7888) (K. Y. Lo). The authors also thank the Center for Emerging Material
and Advanced Devices, National Taiwan University, for the use of the cell
culture room.
DISCLOSURES:
The
authors have nothing to disclose.
REFERENCES
4 Adler, J. Chemoreceptors in bacteria. Science 166, 1588-1597 (1969).
15 Lin, F. et al. Lymphocyte electrotaxis in vitro and in vivo. J Immunol 181, 2465-2471 (2008).