Supporting Information
Transfer printing of transfected cell microarrays from poly(ethylene glycol)-oleyl
surfaces onto biological hydrogels
Satoshi Yamaguchi1, Senori Komiya2, Erika Matsunuma1, Shinya Yamahira2, Takanori
Kihara3, Jun Miyake3 and Teruyuki Nagamune1,2*
1
Department of Chemistry and Biotechnology, Graduate School of Engineering
2
Department of Bioengineering, Graduate School of Engineering, The University of
Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
3
Department of Mechanical Science and Bioengineering, Graduate School of
Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka
560-8531, Japan
S1
Materials and Methods
Experimental
PEG-lipid coating and spotting
PEG-oleyl surfaces were prepared via an amide-coupling reaction between PEG-oleyl
with an N-hydroxysuccinimidyl (NHS) ester, 1 (Fig. 1A; a gift from NOF corporation,
Tokyo, Japan) and albumin-coated surfaces, as previously reported.11,12 To coat the
whole surface of glass slides (7.6 cm × 2.6 cm; Matsunami Glass, Kishiwada, Japan), 7
mL of 0.1 mM 1 in PBS was placed on glass slides coated with bovine serum albumin
(BSA) and incubated for 30 min. The dotted pattern of PEG-oleyl surfaces was prepared
on the BSA-coated glass slides by spotting 20 nL of 0.01 mM 1 in PBS with an inkjet
printer (from Kubota Comps Corporation, Amagasaki, Japan).
Preparation of the surfaces for reverse transfection
Transfection mixture solutions were prepared by mixing 0–20 pmol of siRNA, 1 μL of
Lipofectamine™ 2000 (LF2000; Invitrogen, Carlsbad, CA, USA), 15.5 μL of DMEM
(Nissui Pharmaceutical, Tokyo, Japan), 5 μL of 4 mg/mL fibronectin (Life Laboratory,
Yamagata, Japan) and 2.5 μL of 0.1% (w/v) gelatin. Anti-EGFP, anti-matrix
metalloproteinase-14 (MMP-14) and anti-paxillin (PXN) siRNAs were purchased from
Qiagen (Venlo, the Netherlands). After pre-incubation for 20 min, 20 nL of each
transfection mixture solution was spotted onto the surfaces evenly coated with
PEG-oleyl or those with the dotted pattern of PEG-oleyl using an inkjet printer as
described above. In the latter case, the transfection mixture solutions were spotted on
the PEG-oleyl spots.
S2
Preparation of hydrogel sheets
A collagen gel sheet was prepared on a porous polycarbonate membrane with a nylon
frame as previously reported.11 A nylon frame (inside: 1.4 cm × 1.4 cm, outside: 1.8 cm
× 1.8 cm) was adhered to a polycarbonate porous membrane (17 cm × 17 cm; Whatman,
Clifton, NJ). The solution of collagen type І (Nitta Gelatin, Osaka, Japan) was cast into
the nylon frame (approximately 180 μL) with cooling on a metallic plate, which was
chilled at 4°C. The collagen solution was turned into a gel by incubating at 37°C under
5% CO2 for 30–60 min.
Preparation of cancer cells stably expressing EGFP
The gene encoding enhanced green fluorescent protein (EGFP) was obtained by
digesting pEGFP-N1 (Clontech Laboratories, Inc., Mountain View, CA, USA) with
XhoI and NotI, and inserted into the pMXs-IRES-Puro retroviral vector (pMXs-IP; Cell
Biolabs, Inc., San Diego, CA, USA), resulting in pMXs-EGFP-IP. The retroviral
packaging
cell
line,
Plat-E,
was
transfected
with
pMXs-EGFP-IP
using
Lipofectamine™ LTX (Invitrogen, Carlsbad, CA, USA). The culture supernatant on day
2 was used for retroviral transduction of the human tumor cell lines, HeLa and HT-1080,
using RetroNectin (Takara Bio Inc., Otsu, Japan). Sorting of EGFP-positive HeLa and
HT-1080 transductants was performed with a FACSCalibur flow cytometer
(Becton-Dickinson, Lexington, KY, USA) with excitation at 488 nm and fluorescence
detection at 530 ± 15 nm in a single cell. These EGFP-positive HeLa and HT-1080 cells
were named HeLa-EGFP and HT-1080-EGFP, respectively.
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Image analysis of cell-stamped or cell-embedded hydrogel sheets
Fluorescent images of the microarrays of transfected cells were obtained by scanning
with a Typhoon 9400 Scanner (GE Healthcare, Little Chalfont, UK) using the excitation
lasers at 488 nm for imaging EGFP-expressing cells. For evaluating the knockdown
effect of transfection of anti-EGFP siRNA, the fluorescent images of the HeLa-EGFP
cells were obtained after incubation (37°C, 5% CO2) for 0, 22 and 66 h. The fluorescent
images at 66 h were analyzed using ImageJ software (NIH), and the decrease in
fluorescence compared with that of the non-transfected area was quantified by obtaining
the relative mean whiteness of the spot images. To evaluate the invasion of transfected
cancer cells, the fluorescent images of the transfected HT-1080-EGFP cells were
obtained after incubation (37°C, 5% CO2) for 0, 12 and 24 h. The relative spot areas
compared with the spot area at 0 h were calculated by image analysis using the
make-binary mode in ImageJ.
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Supplementary Figure
Fig. S1
Visualization of the expansion of cell clusters on cell microarrays in a 3D
collagen gel. (A) The scheme for visualizing cancer cell invasion on the stamped cell
microarray in a collagen gel sheet. (B, C) Fluorescent images of microarrays of
HeLa-EGFP cells (B) and HT-1080-EGFP cells (C) embedded in the collagen gel sheets
were obtained with a fluorescent image scanner using the excitation lasers at 488 nm
before and after incubation for 24 h. Scale bars are 1 mm.
S5