SUPPLEMENTARY INFORMATION I
Fabrication of nanopatterned substrates
We developed a method for the fabrication of large area uniform nanopatterns with
different sets of geometrical parameters, each one available in several copies. We employed a
single or a double step thermal Nanoimprint Lithography (NIL) and plasma etching to
fabricate gratings of lines and arrays of dots starting from a few primary masters. The final
substrates for the cultures were obtained by curing a soft PDMS elastomer sandwiched
between glass slides and a silicon master fabricated in the previous step.
Master fabrication
Commercial gratings of lines (250 and 500 nm of width and period, respectively)
produced by Interference Lithography (IL) and etched into silicon to a depth of 320 nm were
purchased from Amo GmbH.
Silicon moulds with depth of 50 to 400 nm and containing different types of patterns
(gratings and arrays of pillars and holes), were obtained from the primary gratings by single
or double step Nanoimprinting Lithography (NIL) and plasma etching. The double step NIL
process developed to this purpose, enables Boolean operations of union (U) and intersection
(∩) of patterns. A bi-layer of two stacked NIL resists with different glass transition
temperatures is used, the top one acting as imaging layer (i.e. the one into which the
nanostructures of the master are indented) and the bottom one to which the pattern of the top
resist layer is transferred by oxygen plasma; repeating the cycle of steps of NIL, pattern
transfer into the bottom resist by oxygen plasma, and thermal re-flow of the top polymer layer
results in the implementation of the Boolean operators of union of patterns. Since every step
requires plasma etching to transfer the pattern to the bottom layer, the total thickness of the
top resist layer is progressively reduced; therefore the initial top layer thickness is chosen in
such a way that enough material is present for all subsequent NIL steps.
Fabrication of soft PDMS substrates. Substrates for cell culture were obtained by mold
casting of Sylgard 184 PDMS elastomer (Dow Corning, Midland, MI). The latter consists of a
base and a curing agent, typically mixed at 1:10 (w) ratio and degassed under vacuum for at
least 30 minutes. 1.5x1.5 cm2 silicon master with the selected topography were used as molds.
The stack of glass/PDMS/mould was kept at 150 kPa of pressure and the temperature was
raised to 120°C for 1h for curing. After cooling the system to RT, the PDMS substrates were
peeled off from the masters.
SUPPLEMENTARY INFORMATION 2:
Substrate characterization: Atomic Force Microscopy (AFM) of HEMA hydrophilized
nanopatterned substrate was performed using a Nanowizard II AFM (JPK, Berlin, Germany)
combined with an inverted optical microscope (Zeiss Axiovert 200, Göttingen, Germany).
The effective elastic modulus of substrates was measured by AFM force spectroscopy. A 5
m diameter Si bead was attached at the end of a tipless cantilever using a UV-curable glue.
Silicon nitride cantilevers were V-shaped, 100 m long and with a nominal spring constant of
0.32 N/m from NANOWORLD (PNP-TR-TL-20)
The cantilever spring constants were calibrated individually after bead attachment. PDMS
substrates were made hydrophilic, coated with laminin and immersed in a buffer solution
(PBS 1%) to reduce the adhesion with the probe. The loading force was adjusted to reach an
indentation of 20% of the height of the nanostructures. Around 1000 force-distance curves
were acquired for each substrate in different areas and analyzed with JPK Image Processing
software (Berlin, Germany). Each curve was fitted with the Hertz model (Rosenbluth et al.,
2006) to calculate the effective sample elasticity, which results from a combination of pillar
bending, shearing, dislocation and compression effects (Schoen et al., 2010). The size of the
beads allows the measurement of an average effect rather than the response of a single pillar.
We believe that this is a reasonable approximation of the cell substrate interaction, at least in a
first stage of cellular adhesion and development.
SUPPLEMENTRAY INFORMATION 3:
Embryonic Stem Cells culture and Stromal Cell-Derived Inducing Activity (SDIA)
protocol. ES cells were induced to differentiate into neurons as previously described (Ban et
al. 2007). BF1/lacZ ES cells were plated on Mitomycin C (Sigma-Aldrich, St.Louis, MO)
treated-MS5 cells as single-cell suspensions at a density of 250 cells/cm2 in Knockout Serum
Replacement (KSR, Invitrogen, Carlsbad, CA) medium and cultured for 6 days changing
medium every day. The ES-derived epithelial structure that developed under these conditions
was incubated with Hank’s balanced salt solution (HBSS, Sigma-Aldrich, St.Louis, MO)
containing 0.15 M Hepes (Sigma-Aldrich, St.Louis, MO) for 15 min and mechanically
separated from the monolayer by flushing through the pipette tip near the colony without
removing the feeder. The detached colonies were resuspended with KSR medium and plated
on 15 µg/ml polyornithine (Sigma-Aldrich, St.Louis, MO) - 1µg/ml fibronectin (Invitrogen,
Carlsbad, CA) coated dishes. After 2-3 hours the medium was replaced with amplification
medium: N2 medium containing 10ng/ml basic fibroblast growth factor (bFGF, R&D
Systems, Minneapolis, MN) and 1µg/ml fibronectin. Neuronal precursors were induced to
proliferate in the presence of bFGF for 4 days and then mechanically dissociated in HBSS
solution, resuspended in N2 medium and then plated on 15 µg/ml polyornithine-1 µg/ml
laminin-coated PDMS substrates at a density of 3x104 cells/cm2. Removal of bFGF induces
differentiation and cells were analyzed 48 hours after induction, unless otherwise specified.
For the evaluation of the substrate effect on the neuronal differentiation, neuronal precursors
derived from the same ES cell culture were plated on a complete set of substrates. For each
validation we repeated at least 4 times the complete experiment.
SUPPLEMENTARY INFORMATION 4:
Immunocytochemistry The whole assay was performed at RT. Cells were fixed in 4%
paraformaldehyde (Sigma-Aldrich, St.Louis, MO)-0.15% picric acid (Sigma-Aldrich,
St.Louis, MO) in phosphate-buffered saline (PBS, Invitrogen, Carlsbad, CA) for 20 min.
Sample were then saturated with 0.1 M glycine (Sigma-Aldrich, St.Louis, MO) for 5 min,
permeabilized with 0.1% triton X-100 (Sigma-Aldrich, St.Louis, MO) for 5 min and blocked
with 0.5% BSA (Sigma-Aldrich, St.Louis, MO) in PBS for 30 min. Primary antibody was
then incubated for 1h. Primary antibodies used: mouse monoclonal antibody against neuronal
class III β-tubulin - Tuj1 (Covance, Berkeley, CA); mouse monoclonal antibody anti-nestin
(Chemicon International). The secondary antibodies were: goat anti-mouse immunoglobulin
(Ig) G1-FITC and IgG2a-TRITC (Southern Biotech, Birmingham, AL). Total nuclei were
stained with 2 µg/ml Hoechst 33342 (Sigma-Aldrich, St.Louis, MO) in PBS for 5 minutes.
Immunofluorescence (IF) images were acquired using a Zeiss Axioskop 2 MOT fluorescence
microscope with a Zeiss plan-NEOFLUAR 20x objective. Random images were acquired on
each substrate to cover approximately the 60% of the whole area. The cell number and
neurites length were analysed with a program for imaging processing and analysis (ImageJ)
and the NeuronJ plugin. To increase the statistical significance of the effect of the substrate on
the neuronal differentiation, at least 1000 cells per coverslip were counted.
To qualitatively evaluate the laminin adsorption, PDMS substrates were coated with 15 µg/ml
polyornithine and 2 µg/ml laminin , fixed with 4% paraformaldehyde - 0.15% picric acid in
PBS for 20 min, saturated with 0.1 M glycine for 5 min, incubated with mouse monoclonal
anti-laminin antibody (Sigma-Aldrich, St.Louis, MO) for 1h followed by 30 min incubation
with goat anti-mouse 594 Alexa antibody (Invitrogen, Carlsbad, CA). All the incubations were
performed at room temperature (20-22°C). The samples were examined using a Leica DM6000
fluorescent microscope equipped with DIC and fluorescence optics, CCD camera and Volocity 5.4 3D
imaging software (PerkinElmer, Coventry, UK). The fluorescence images were collected with a 63x
magnification and 1.4 NA oil-immersion objective or with 100x magnification and 1.3 NA oilimmersion objective. For each image the same exposure and gain settings were used. The images were
deconvolved using the Volocity Restoration. ImageJ software was used to extract an intensity plot
profile inside an area. Areas 40×10µm were analysed on flat and nanopatterned PDMS.
REFERENCES:
Ban J, Bonifazi P, Pinato G, Broccard FD, Studer L, Torre V, Ruaro ME. 2007. Embryonic
stem cell-derived neurons form functional networks in vitro. Stem Cells 25:738–749.
Rosenbluth MJ, Lam WA, Fletcher DA. 2006. Force microscopy of nonadherent cells: a
comparison of leukemia cell deformability. Biophys. J. 90:2994–3003.
Schoen I, Hu W, Klotzsch E, Vogel V. 2010. Probing Cellular Traction Forces by Micropillar
Arrays: Contribution of Substrate Warping to Pillar Deflection. Nano Lett 10:1823–1830.