Supporting Information
Nephrocyte-Neurocyte Interaction and Cellular Metabolic Analysis on Membrane-integrated
Microfluidic Device
Qichen Zhuang1,2, Shiqi Wang1, Jie Zhang1, Ziyi He1, Haifang Li1, Yuan Ma1, 2, * & Jin-Ming
Lin1, 2*
1
Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation,
Tsinghua University, Beijing 100084, China
2
The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University,
Beijing 100084, China
* Corresponding authors. E-mail: jmlin@mail.tsinghua.edu.cn (J.-M. Lin); mayuan@tsinghua.edu.cn (Y.
Ma)
1
Fabrication of the Membrane-Integrated Microfluidic Device
The membrane-integrated microfluidic device was fabricated by standard soft lithographic and replica
molding techniques. Briefly, PDMS prepolymer was mixed with curing agent at a weight ratio of 10:1. The
mixture was then cast onto a 75mm silicon wafer having positive relief patterns of microchannels and then
cured at 65 °C for more than 3 h. The mold was fabricated by spin-coating photoresist (SU-8 2050) twice at
1000 rpm for 60 s. The cured top layer of the microfluidic chip with four parallel microchannels was peeled
from the silicon wafer and then the top layer was punched to create connection holes. The bottom layer was
digged by a iron tube on a cured smooth PDMS replica and then irreversibly sealed with a glass slide by
oxygen plasma (PDC-32G, Harrick Plasma, Ithaca, NY, USA) treatment. Then, the top layer, the
polycarbonate (PC) porous membrane and the bottom layer would be sealed together by PDMS mortar
layers.
To create PDMS mortar layers, PDMS prepolymer described above was spin-coated on a clean silicon
wafer for 60 s at 3000 rpm to generate a thin mortar layer. The top and bottom layers of the microfluidic
chip were placed onto the silicon wafer spin-coated with adhesive PDMS mortar and allowed to stay in
contact for at least 30 s. Then, the PC membrane was placed onto the bottom layer of the microfluidic chip
with adhesive PDMS mortar using tweezer. The PC membranes have 5 μm diameter pores, which are small
enough to prevent cell migration and large enough to permit molecular transport. The bottom piece with the
membrane attached was then aligned and bonded with the top piece. The combined device was placed at
room temperature overnight and then 65℃ for an hour.
The dimensions of the channels on the top layer were 16 mm long, 800 μm wide and 230 μm deep,
while the dimensions of the channel on the bottom layer were 20 mm long, 8 mm wide. The relationship
between the height of the bottom channel (H) and the mass of the prepolymer (m) was shown in Figure S4.
Cell Culture
The PC12 cells (Cancer Institute & Hospital of the Chinese Academy of Medical Science, Beijing,
China) were maintained in RPMI 1640 with L-glutamine supplemented with 10% fetal bovine serum (FBS),
15% horse serum (HS), 100 units/mL penicillin, and 100 units/mL streptomycin in a humidified
atmosphere of 95% air and 5% CO2 at 37 ℃. The 293 cells (Cancer Institute & Hospital of the Chinese
Academy of Medical Science, Beijing, China) were maintained in Dulbecco’s modified Eagle’s minimal
essential medium (DMEM) with high glucose supplemented with 10% fetal bovine serum (FBS), 100
units/mL penicillin, and 100 units/mL streptomycin in a humidified atmosphere of 95% air and 5% CO 2 at
37°C. The PC12 and 293 cells were maintained in culture flasks for 2-3 days prior to commencing the
microfluidic experiments. All the experiments were carried out when the cells were in the exponential
growth phase.
2
Channel Modification for Cell Adhesion and Growth
Four reagents, 0.1% PLL (30kD-70kD), 0.1% PLL (70kD-150kD), 0.1% gelatin and FBS were used to
modify the glass substrate and the PC membrane for cell adhesion and growth. Before modification, the
membrane-integrated microfluidic devices were sterilized under UV light for 1 h. Then, modification
solution was injected into the channel (top and bottom channels respectively) and incubated for at least 6 h
to modify the glass substrate and the PC membrane. The device was then rinsed with PBS buffer three
times, and dried at room temperature in a super clean bench.
About 400 μL PC12 cell suspension at a density of 1×106 cells/mL was injected into the inlet of the
bottom channel, and a negative pressure was generated at the outlet by a pipette to allow the cells to fill the
bottom channel. In this step, the PC12 cells were suspended in RPMI 1640 supplemented with 150 ng/mL
mNGF. The cell culture medium was then gently injected into the top channels, and the inlets and outlets
were covered with culture medium in order to avoid the evaporation. The devices were then put into a cell
culture plate and finally placed in a 37 ℃ humidified incubator with 5% CO2 for long-term culture. The
medium was replaced at Day 1, 3 and 5 to supply enough nutrients and to wash away the cellular debris and
waste. Then the cells were characterized with Calcein AM/EthD-1 (Live/Dead Viability/Cytotoxicity Kit;
Invitrogen, Carlsbad, CA, USA) and imaged by the Leica DMI 4000B (Wetzlar, Germany) fluorescence
microscopy for qualitative assessment of cell spreading and cell viability. Three random images were taken
per device as a representative of the full coverage of cells and three independent samples (n=3) were tested.
On-Chip Treatment of FBS and mNGF for PC12 Cell Differentiation
Before the differentiated PC12 cells and 293 cells co-cultured on the microchip, we optimized the
concentration of FBS and mNGF in the medium.
The PC12 cells were supplemented with 150 ng/mL mNGF with the concentration of FBS in four
experimental groups being 10%, 5%, 1% and 0%, respectively. And the undifferentiated PC12 cells
maintained in medium supplemented with 10% fetal bovine serum (FBS) and 15% horse serum (HS), was
set as control group. Then, the cell culture medium was then gently injected into the top channels, and the
inlets and outlets were covered with fresh medium in order to avoid the evaporation. The devices were then
put into a cell culture plate and finally placed in a 37 ℃ humidified incubator with 5% CO2 for long-term
culture. The medium was replaced after the first day of culture, and then the medium was changed every 48
h to supply enough nutrients and to wash away the cellular debris and waste. The cells were cultured for 5
days and then we carried out a cytoskeleton analysis, a protein expression analysis and a cell viability
analysis to evaluate cell differentiation and cell viability.
To determine the neural cytoskeletal construction, cultures were stained with Phalloidin CruzFluor™
633 Conjugate to reveal clearly the cell cytoskeleton. After the PC12 cells were cultured with different
concentration of FBS for 5 days by NGF stimulated, each specimen was rinsed with PBS to remove any
unattached cells. For fluorescent microscopy observation, the cells were fixed in situ for 10 min in
3
immunol staining fix solution (Beyotime, Inc., Nantong, China). Thereafter, filamentous actin was stained
with Phalloidin CruzFluor™ 633 Conjugate, whereas the nuclei were stained with DAPI. Subsequently, the
specimens were examined using a confocal laser scanning microscope (LSM710META, Zeiss).
To determine the expression of microtubule-associated protein 2 (MAP-2), the cells were stained with
MAP-2 (A-4): sc-74421 to reveal clearly the expression of MAP-2 by the differentiated PC12 cells. After
the PC12 cells were cultured with different concentration of FBS for 5 days by NGF stimulated, each
specimen was rinsed with PBS to remove any unattached cells. For fluorescent microscopy observation, the
cells were fixed in situ for 10 min in immunol staining fix solution. Thereafter, each specimen was blocked
by the immunol staining blocking buffer (Beyotime, Inc., Nantong, China) for 3 h and then MAP-2 in the
differentiated PC12 cells were stained with MAP-2 (A-4): sc-74421 overnight. Then each specimen was
rinsed with PBS and then Cy3-labeled Goat Anti-Mouse IgG (H+L) was added into each specimen for 30
min, whereas the nuclei were stained with DAPI. Subsequently, the specimens were examined using a
confocal laser scanning microscope (LSM710META, Zeiss).
To determine the cell viability in each specimen with different concentration of FBS, we carried out
CCK-8 tests for each specimen on Day1, Day3 and Day 5 respectively. Incubate 3 plates cell suspension
(200 µL/well) with different concentration of FBS (experimental group) and control group in a 96-well
plate for 5 Days, respectively. On Day 1, add 20 µL of the CCK-8 solution to each well of Plate 1, and then
incubate the plate for 3 hours in the incubator. Thereafter, measure the absorbance at 450 nm using a
microplate reader. And then repeated these operations for Plate 2 and Plate 3 on Day 3 and Day 5
respectively.
To optimize the concentration of mNGF for inducing PC12 cells differentiated, we carried out a cell
viability analysis to evaluate the differentiated PC12 cells viability in culture medium with 0 ng/mL, 10
ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, 150 ng/mL and 200 ng/mL on Day1, Day3 and Day5,
respectively.
MS Detection
The Thermo LTQ mass spectrometer parameters were as followed: capillary temperature = 275 ℃,
capillary voltage = 9 V, tube lens voltage = 100 V, max injection time = 200 ms, micro scans = 3, spray
voltage = +2000 V. The collision energy used in tandem mass spectrometric analysis was 30 for MS2 and
50 for MS3. Borosilicate glass capillaries (I.D. = 0.6 mm, O.D. = 1 mm, Vital Sense Scientific Instruments
Co. Ltd.) were pulled by P-2000 (Sutter Instrument Co.) to make the emitters (I.D. of the tip is 1 μm) for
Nano-ESI. The parameters of P-2000 were as followed: Heat = 300, FIL = 5, VEL = 40, DEL = 140, PUL
= 60.
4
Figure S1. Channel modification for cell adhesion and growth on the microchip. The microfluidic channels
were modified by (A) PLL, 30kD-70kD; (B) PLL, 70kD-150kD; (C) Gelatin; (D) Fetal Bovine Serum
(FBS). Scale bar: 100 μm.
5
Figure S2. Epinephrine detection by the Nano-ESI-MS. (A) Full scan spectrum of 20 ng/mL standard
epinephrine. (B) The MS2 spectrum of 184.1 ([M+H]+) of 20 ng/mL standard epinephrine. (C) The MS3
spectrum of 166.1 ([M-H2O+H]+) of 20 ng/mL standard epinephrine. (D) The MS3 spectrum of 166.1
([M-H2O+H]+) of 20 ng/mL standard epinephrine in RPMI 1640 culture medium. Spectra of epinephrine
were obtained in the positive-ion mode.
6
Figure S3. Epinephrine detection by the Nano-ESI-MS. (A) The MS3 spectrum of 166.1 ([M-H2O+H]+) of
the cellular metabolite of differentiated PC12 cells co-cultured with 293 cells in a 12-well transwell plate.
(B) The MS3 spectrum of 166.1 ([M-H2O+H]+) of 1640 culture medium with 5% FBS，150 ng/mL mNGF
and 2.2 mM CaCl2. (C) The MS3 spectrum of 166.1 ([M-H2O+H]+) of the cellular metabolite of
undifferentiated PC12 cells cultured alone. (D) The MS3 spectrum of 166.1 ([M-H2O+H]+) of the cellular
metabolite of differentiated PC12 cells cultured alone. (E) The MS3 spectrum of 166.1 ([M-H2O+H]+) of
the cellular metabolite of 293 cells cultured alone. (F) The MS3 spectrum of 166.1 ([M-H2O+H]+) of the
cellular metabolite of undifferentiated PC12 cells co-cultured with 293 cells. Spectra of epinephrine were
obtained in the positive-ion mode.
7
Figure S4. Relationship between the height of the bottom channel (H) and the mass of the prepolymer (m).
8
Table S1. t-test of the F-actin morphology assay
p-value
Control
Control
0% FBS
1% FBS
5% FBS
10% FBS
7.02×10-2
5.97×10-1
5.08×10-1
4.43×10-4
2.89×10-2
2.10×10-1
3.34×10-5
6.37×10-1
1.89×10-3
0% FBS
1% FBS
3.14×10-1
5% FBS
Table S2. t-test of the MAP-2 expression assay
p-value
Control
Control
0% FBS
1% FBS
5% FBS
10% FBS
5.56×10-7
9.88×10-14
9.02×10-12
9.60×10-11
3.38×10-1
5.78×10-5
1.17×10-2
2.20×10-5
1.72×10-2
0% FBS
1% FBS
1.15×10-2
5% FBS
9