Fabrication and characterization of protein arrays for stem cell patterning

Fabrication and characterization of protein arrays for stem cell patterning

Laura Ceriotti,a Leonora Buzanska,bc Hubert Rauscher,a Ilaria Mannelli,a Lucel Sirghi,a Douglas Gilliland,a

Marina Hasiwa,b Frederic Bretagnol,a M. Zychowicz,ac Ana Ruiz,a Susanne Bremer,b Sandra Coecke,b

Pascal Colpo*a and Francois Rossia

Received 21st August 2008, Accepted 11th December 2008

First published as an Advance Article on the web 6th February 2009

DOI: 10.1039/b814616k

Microarrays of fibronectin and other extracellular matrix (ECM) proteins were fabricated on plasma-

deposited poly(ethyleneoxide) (PEO-like) film coated glass slides to study adhesion of stem cells. The

arrays were generated by using a non-contact printing technology. The stability and the quality of the

spots of fibronectin, used as protein model, were assessed by time of flight secondary ion mass

spectrometry (ToF-SIMS), ellipsometry and atomic force microscopy (AFM). It was found that

saturation with a mass density of 112 ! 4 ng/cm2 is reached when protein solutions at concentrations

higher than 84 mg/ml are spotted. Fibronectin on the surface form a uniform sub-monolayer with

a surface coverage that depends on the spotting solution concentration, as qualitatively demonstrated

by AFMmeasurements. The active conformation of the spotted fibronectin was verified by performing

an immunoassay with antibodies specific for the fibronectin RGD sequence by surface plasmon

resonance (SPR) imaging. An immunorecognition efficiency of up to 22% was found for a spot with 3%

coverage as estimated by ellipsometry. Human umbilical cord blood neural stem cells (HUCB-NSCs)

were cultured on different ECM proteins (fibronectin, laminin, collagen I, collagen III and collagen V)

arrays and showed protein concentration dependent adhesion on the micro-spots. The cell nuclei were

stained for cell counting and preliminary specific cell staining was performed to evaluate the

differentiation stage of HUCB-NSCs on such spots. The array platform developed in this study

provides a promising approach to investigate in high throughput manner how surfaces patterned

with extracellular matrix (ECM) proteins influence stem cell adhesion and development.

1. Introduction

Surfaces engineered in order to create micro patterns with areas

that either promote or prevent cell adhesion provide an impor-

tant tool for studying the factors that affect cell adhesion,

migration and proliferation as well as cell–cell and cell–surface

interactions.1,2 For example, the commitment of stem cells to

different specific lineages depends on the cell shape which is

defined strongly by the microenvironment (including the

substrate) where the cells grow.1 Besides, micro-patterned

surfaces were shown to be valuable platforms for the develop-

ment of miniaturized systems, such as microfluidic platforms for

long-term cell culture studies,3 toxicity screening in two-dimen-

sional co-culture systems4 and cell based arrays for high

throughput analysis of cellular functions.5–8 Cell microarrays

represent, in fact, a promising alternative to microwell plates for

studying cell adhesion on a large variety of materials, in partic-

ular when only a limited number of cells is available.9

Many methodologies for cell patterning have already been

described in the literature and include stencil assisted patterning,

photolithography and soft lithography (ref. 1, 10 and references

within). Ablation of anti-adhesive polymers,11 photocleavage of

protecting groups12 or functionalization of cell repellent poly-

meric materials by plasma13,14 or UV light7,15 are also methods

used to create patterned surfaces for promoting cell adhesion.

An interesting approach to create cell arrays is to pattern

proteins5 or polymers8 onto surfaces previously coated with cell

repellent polymeric films by robotic spotting technology. The

spotting can be performed using pin (contact) or piezoelectric

(non contact) depositions to create spots of biomolecules, to

which the cells can adhere and grow forming spatially confined

and separated islands.16,17 Such cell array platforms developed

on various kinds of treated surfaces were successfully used in

different studies.5–8

In this paper we report on the optimization of protein

microarrays generated by piezoelectric printing technology on

a plasma deposited PEO-like coating used as protein and cell

repellent layer.18 Unlike the covalent attachment of PEG on

glass slides based on silanization protocols using PEG-silane

solutions19 or mediated by silane and crosslinkers,17 the plasma

deposition of the polymer is fast and can be performed on a wide

range of materials, e.g. glass and plastics, either on flat as well as

on 3D substrates, e.g. Petri dishes or chambers.14,20 Further-

more, in contrast to their non adhesive properties in liquid

environment, the PEO-like films accept protein immobilization

aNanotechnology and Molecular Imaging Unit, European Commission,Joint Research Centre, Institute for Health and Consumer Protection, viaE. Fermi 2749, 21027 Ispra, VA, Italy. E-mail: [email protected]; Fax:+0039 0332 785787bEuropean Centre for the Validation of Alternative Methods, EuropeanCommission, Joint Research Centre, Institute for Health and ConsumerProtection, via E. Fermi 2749, 21027 Ispra, VA, ItalycMedical Research Centre, Polish Academy of Sciences, PawinskiegoStreet 5, 02-106 Warsaw, Poland

1406 | Soft Matter, 2009, 5, 1406–1416 This journal is ª The Royal Society of Chemistry 2009

PAPER www.rsc.org/softmatter | Soft Matter

when printed in dry conditions, e.g. by microcontact printing

(MC).21–23 The piezoelectric printing technique offers the

opportunity to easily use different array layouts, spot different

materials at various concentrations in series and dispense precise

volumes of solution without carryover of probe from one spot to

another, thereby generating spots of highly reproducible protein

content.

In this work, the printing protocol for the generation of ECM

protein arrays has been developed and optimized in terms of

spot size and array layout using fibronectin as protein model.

The fibronectin spots on PEO-like film have been characterized

qualitatively and quantitatively by time of flight secondary ion

mass spectrometry (ToF-SIMS), ellipsometry and atomic force

microscopy (AFM). Furthermore, the accessibility of the fibro-

nectin RGD peptides, which interact with the integrins of the

cellular membrane and are involved in the transduction of the

outside-in signaling, was assessed by surface plasmon resonance

(SPR) imaging and confirmed by cell culture experiments.

Human umbilical cord blood neural stem cells (HUCB-NSCs)24

were used to verify the array stability in cell culture conditions

and the effect of different ECM protein types and concentra-

tions on cell adhesion. A scheme of the processing steps used for

the fabrication of the protein/cell microarrays is presented in

Fig. 1.

2. Experimental

2.1. Substrate preparation

Standard glass slides (obtained from Sigma) were rinsed in

ethanol before PEO-like coating deposition. To improve the

polymer adhesion on glass, the slides were coated with a thin

layer of SiOx. The SiOx coating was obtained by Plasma

Enhanced Chemical Vapour (PE-CVD) deposition using an

inductively coupled plasma source (ICP) and a hex-

adimethylsiloxane precursor (Sigma Aldrich) mixed with O2 and

Ar (working pressure 50 mTorr, ICP power ¼ 450 W). The

deposition time was 5 min for a thickness of 500 nm.

2.2. PEO-like coating deposition

The system used for the plasma-polymerized PEO-like film

deposition was a homemade reactor of stainless-steel (300 mm #

300 mm # 150 mm) with two symmetrical internal parallel-plate

electrodes (diameter of the electrodes ¼ 140 mm, distance

between the two electrodes ¼ 50 mm).18 The plasma was gener-

ated by a radio frequency generator (13.56 MHz) connected to

the upper electrode whereas the other electrode was grounded

and used as a sample holder. The plasma polymerization was

carried out by using a pulsed plasma discharge (time on¼ 10 ms,

time off ¼ 100 ms, nominal power ¼ 5 W) of pure diethylene

glycol dimethyl ether [DEGDME, (CH3OCH2CH2)2O] vapours

(from Sigma–Aldrich, used as received). The working pressure

was kept constant at 2.7 Pa (50 mTorr). More details can be

found in Bretagnol et al.18 For the cell culture experiments,

a 20 nm thick PEO-like film was deposited on microscope glass

slides cut in half. For the SPR imaging analysis, the film

deposited on the SPR slide was thinner than 20 nm in order to

remain in the thickness range usable by the instrument. Ellips-

ometry measurements of the same PEO-like film deposited on

silicon gave a film thickness of 11.5 nm.

2.3. Piezoelectric dispenser

Spotting of the proteins on the substrates was performed with

the S3 sciFLEXARRAYER (Scienion AG, Germany) using the

piezoelectric printing technology. The instrument has a three-

axis micro-positioning system (accuracy 10 mm) and is equipped

with a glass nozzle 80 mm in diameter. A stroboscopic camera

allows visual monitoring to adjust piezo voltages and pulse

durations for reliable droplet ejection and to avoid satellite

drops. Single drops ejected from the nozzle have a volume of

400 pL under our experimental conditions. In our study we

dispensed between 1- and 5-drop volumes. The vertical sepa-

ration between the nozzle and the substrate was typically

0.5 mm. The distance between spots can vary but typically for

cell culture experiments a pitch of 600 mm was used. Arrays of

Fig. 1 A scheme of the steps involved in the present work. After PEO-like film deposition by plasma, protein solutions are spotted on the coated slides

by piezo electric printing technology. The patterned slides are then characterized or used as substrate for cell culture experiments.

This journal is ª The Royal Society of Chemistry 2009 Soft Matter, 2009, 5, 1406–1416 | 1407

10 # 10 spots arranged in 2 lines of 3 arrays each were prepared

on half slides.

2.4. Protein spotting

Fibronectin (from Sigma, F0895) was purchased in Tris buffer at

pH 7.5. The protein was first spotted in phosphate buffered saline

(PBS, pH 7.4)(Sigma). Then, the fibronectin was diluted in

a printing buffer to better control spot size and evaporation. The

selected printing buffer consists of 100 mM acetate (Riedel

deHa€en), pH 5, 5 mM EDTA (Merck), 0.01% Triton-X 100

(Fluka) and 0.1% glycerol (Carlo Erba). In order to assess the

effect of the printing buffer alone on cell adhesion, the printing

buffer was also spotted on the PEO-like film. Laminin (L6274),

collagen I (C7774), collagen III (C4407), collagen V (C3657),

bovine serum albumin (BSA)(A4503) and poly-L-lysin

(PLL)(P4707) were also purchased from Sigma and spotted in

the printing buffer on the PEO-like coating. The spotted samples

were inspected with an optical microscope to confirm their

quality (circularity and dimensions) and then stored at 4 $C in

wet conditions5 until further use, which occurred typically within

one week after slide preparation. Longer storage times were not

considered. Typically, the patterned substrates were exposed to

ultraviolet radiation for 10 min and then rinsed in PBS before cell

culturing. It was found that for relatively short culture experi-

ments (1–2 weeks) the UV treatment can be omitted since the

cells did not show any contamination problem.

2.5. Time of flight secondary ion mass spectroscopy (ToF-

SIMS)

The characterization by ToF-SIMS was performed using

a ToF-SIMS IV (ION-TOF GmbH, Germany) equipped with

a primary 25 keV Bi ion gun operating either in high current

surface spectroscopy mode or imaging mode (mass resolution

(m/Dm) > 8000, lateral resolution %1–3 mm). The analyses were

conducted using (Bi3)+ ions and static SIMS conditions were

respected with the total ion flux being maintained below 1 # 1013

ions/cm2.

2.6. Ellipsometry

The protein spots on the PEO-like coating were analyzed by

ellipsometry. Fibronectin at different concentrations ranging

from 21 up to 333 mg/ml was spotted on PEO-like coated silicon

slide as a two-drop spot. In this way, fibronectin spots of about

230 mm in diameter were obtained. Before analysis, the spotted

samples were rinsed in water and dried under nitrogen flow.

Although no spot was optically visible on the surface once the

sample was dried, it was possible to identify the spots and to

distinguish between the different protein concentrations by

ellipsometry. Ellipsometric data were acquired with a variable

angle imaging ellipsometer (model EP3 by Nanofilm Surface

Analysis GmbH, Germany). All imaging measurements were

performed in air at room temperature at an angle of incidence

of 42$, using a monochromatized Xe lamp at a wavelength of

l ¼ 554.3 nm as light source. A polarizer-compensator-sample-

analyzer (PCSA) null-ellipsometric procedure was used to obtain

maps of theD andJ angles for the selected area. Thickness maps

are calculated from the D and J maps by point-by-point

modelling with the software EP3View provided with the ellips-

ometer, using a two-layer model with PEO as first layer and the

protein as the second. The thickness (11.5 nm) and the refractive

index of plasma-deposited PEO (nPEO ¼ 1.51) were indepen-

dently determined by an angle-resolved measurement. A refrac-

tive index of 1.46 was used for the protein.25 For the calculation

of the mass density the nominal thickness was averaged over six

equivalent spots deposited from a solution with equal protein

concentration. Based on the Lorentz–Lorenz relation26 for the

refractive index n of a pure substance, the adsorbed mass per

area, i.e. the mass density m per unit area, can be written as27

m ¼Mwd

A

n2 & 1

n2 þ 2

!

(1)

where Mw and A are the molecular weight of the substance and

molar refractivity, respectively, and d is the effective thickness of

the film as determined by ellipsometry. The molar refractivity

of fibronectin was calculated by Thom et al.28 as the sum of the

individual molar refractivities of all amino acids in fibronectin,

using tabulated values.29 Using this approach, a value of

3.99 g/cm3 was obtained for the term Mw/A of fibronectin in dry

state.

2.7. Atomic force microscopy

The AFM measurements were performed with a commercial

atomic force microscope (Smena B from NT-MDT Co.) oper-

ated in tapping mode. For the measurements of the spotted

samples, a commercial AFM probe (NSG 11 from NT-MDT)

with sharpened silicon tip (nominal curvature radius of 10 nm)

was used. The probe was driven at the resonance frequency of its

cantilever with a free amplitude of 100 nm and tapping amplitude

of 50 nm. Before AFM analysis the samples were incubated in

PBS for 1 h, rinsed in deionized water and dried under nitrogen

flow.

2.8. SPR imaging

The immunoreaction experiments on fibronectin were also

monitored with a Surface Plasmon Resonance imaging system

(Genoptics, France). The light source is a collimated LED with

a centre wavelength of 810 nm and a spectral width Dl of about

10 nm. The incident beam is coupled to the surface plasmons by

a prism (Kretschmann configuration).30 For the SPR experi-

ments, the PEO-like film was deposited on a gold covered glass

slide. Then different concentrations of fibronectin (21, 42, 84,

167, 333 mg/ml) were spotted in two rows for each concentration

as 2-drops with 500 mm pitch to form a 10 # 10 array on the

PEO-like coated slide. Then the sample was incubated in 10 mM

PBS for 2 h, rinsed in water, dried and stored at 4 $C until further

use. For the recognition test, the slide was coupled to the prism

by an index matching oil and inserted into the flow chamber

of the SPR system. The reflectivity curve, R(q), is constructed as

a function of the incident angle of the light beam by varying

the angle between 53$ and 63$. The working angle is determined

as the maximum of the first derivative of the R(q) function.

A baseline is recorded in PBS before switching to the protein

flows with the following sequence: (i) 200 mg/ml BSA (Sigma), (ii)

20 mg/ml anti-ovalbumin (Ab-OVA) (Sigma) and (iii) 10 mg/ml


mouse anti-fibronectin monoclonal antibody RGD specific

(Ab-RGD, Chemicon International, MW of 150 kDa). The

reflectivity is monitored in real time during the experiment. The

interaction of the various proteins with the fibronectin spots as

well as with the surrounding PEO-like coating was measured as

reflectance shift with respect to the PBS baseline. The obtained

kinetic curves show the evolution of the protein/antibody inter-

action over time, and the reflectivity difference images immedi-

ately indicate which spots react with the injected sample. The

results, expressed as reflectivity difference with respect to the

baseline DR (%), were calculated as the mean value of signals

from the areas of 6 different spots. Before, between and after

the flow of the individual proteins, PBS was run to condition the

slide surface and to remove the excess of fibronectin molecules

that were not well immobilized on the spots and the proteins

which had not interacted with fibronectin in the spots. The PBS

flow rate used was 12.5 ml/min.

The antibody density on the fibronectin molecules was calcu-

lated by the following formulas:

G ¼DRLzc

SP;R dn=dC

where G is the protein density (ng/cm2), DR the variation of

reflectivity (%), SP,R the variation of reflectivity per unit

of variation of refracted index, Lzc the penetration depth of the

evanescent wave in the medium, dn/dC the variation of the

refractive index as a function of concentration. All the parame-

ters were given by the instrument constructor.

2.9. Cell culture

A neural stem cell line obtained from a non-hematopoietic

fraction of human umbilical cord blood (HUCB-NSC)24 was

maintained as the mixed adherent/floating culture in Dulbecco’s

modified Eagle medium (DMEM/F12, Gibco) containing 2%

fetal bovine serum (FBS, Gibco), insulin-transferrin-selenium

(ITS,1:100, Gibco), antibioticantymycotic solution AAS (1:100,

Gibco), at 37 $C, 5% CO2 and 95% humidity. Serum free medium

contained DMEM/F12, B27 (1:50, Gibco) and EGF (20 ng/ml,

Sigma). HUCB-NSC cultures were propagated every two weeks,

in medium containing 2% serum, by trypsinization (Trypsin/

EDTA 0,025%, Gibco) in 1:4 ratio. For cell array generation, the

cells were plated either at low (1 # 104 cells/cm2) or high (5 # 104

cells/cm2) densities on the spotted slides placed into 5 cm diam-

eter Petri dishes. After cell seeding, the samples were placed into

a cell culture incubator (37 $C and 5% CO2) and agitated for 6 s

every 10 min during the first hour. This period of agitation helps

the distribution of the cells on the protein arrays. Since cells

attached quickly to the spots, the culture medium was replaced

by a fresh one 2 h after cell seeding in order to remove the excess

of non adherent cells and preserve the non-adhesive character of

the PEO-like film. During the course of the experiments (up to

2 weeks) the medium (maintenance medium with 2% of serum or

serum-free) was changed every two days.

2.10. Cell array staining and characterization

During cell culture, the cell arrays were inspected with an Axi-

overt 200 fluorescent microscope (Zeiss). Contrast phase photos

of the arrays were taken every other day during culture. To

obtain an image of a full cell array as presented in this paper for

HUCB-NSCs (Fig. 6) individual photos were taken with the

lowest magnification available (5#) and then combined.

For cell counting experiments, the cells were fixed in 4% par-

aformamide for 20 min after 24 h of culture. For nuclei staining,

the samples were then rinsed in PBS 3 times, immersed in

a solution with Hoestch 33342 (1:1000, Molecular Probes) for

20 min and rinsed extensively in PBS. The slides were either

closed with mounting solution (Fluoromount-G from Southern

Biotech) or kept in PBS for further staining. For the immunos-

taining of differentiating cells, primary antibodies, i.e., mono-

clonal neuronal class III b-tubulin antibody (TUJ1, 1:500,

Covance), and polyclonal rabbit antibodies against S-100b

(1:1000, Swant) were applied overnight. AlexaFluo 488 and

AlexaFluo 546 (1:2000, Jackson) were used to visualize

b-Tubulin III and S-100b, respectively. The counting of the

stained cell nuclei was performed using the WCIF Image

J program.31 Threshold and counter parameters were optimized

for each individual cell spot. Average values and standard

deviation (STD) were calculated for at least 10 spots for each size

and protein concentration from 3 different experiments.

3. Results and discussion

3.1. Protein spots characterization by ToF-SIMS

One of the main prerequisites for the generation of long term

stable biomolecule microarrays is to provide a strong contrast

between well defined active areas (immobilized oligonucleotides,

peptides, proteins, antibodies) and non adhesive zones to avoid

mixing of the biomolecules between different spots. In this work,

the proteins were spotted directly on the PEO-like coated slides.

The stability of the protein array on the PEO-like film and the

effect of the protein concentration on the spot size were evalu-

ated by ToF-SIMS. Different concentrations of fibronectin (21,

42, 84, 167, 333 mg/ml) diluted in PBS were spotted with different

drop numbers (from 1 up to 5 drops) on a coated slide and dried

for 30 min. Then, the sample was incubated overnight in water

prior to analysis. The results are presented in Fig. 2A. These

images show, via the CN(&) signal, that the proteins are present

only on the spots, whereas no proteins are found in between the

spots. This confirms that the proteins remain stably adsorbed on

the PEO-like film after incubation in solution and therefore

can be used as substrate for cell patterning. Furthermore, Fig. 2A

shows that the protein spot sizes depend on the protein

concentration and that the protein molecules are not uniformly

distributed on the spots due to the presence of buffer salts before

rinsing. These salty areas prevent protein adsorption which

results, after rinsing, in dark zones in the ToF-SIMS images.

Since the final objective of the study is to analyze and compare

the behaviour of cells on spots of different kinds of proteins and

protein concentrations, we replaced PBS by a different printing

solution to obtain the same spot size, independent from protein

type and concentration. We tested an acetate buffer at pH

5 which prevents collagen fibril aggregation16 and contains 5 mM

EDTA to prevent laminin polymerization,32 0.01% Triton-X

100 and 0.1% glycerol to slow down the spot evaporation.5

Higher concentrations of the surfactant would compromise the


performance of the piezoelectric spotter due to the creation of

bubbles in the liquid handling unit and nozzle. In this printing

buffer, the spot size remains independent from the protein

concentration as shown in Fig. 2B for the 1 drop fibronectin

spots. It is evident from the CN(&) images that all spots of

fibronectin deposited from 21, 42 and 84 mg/ml solution have the

same diameter of around 200 mm. The same diameter was found

for spots of other ECM proteins, like collagens and laminin (data

not shown). Furthermore, the use of this buffer improved also

the distribution of fibronectin molecules on the spot. For these

reasons, this buffer was used for all further spotting experiments.

3.2. Fibronectin spot density and distribution characterized by

ellipsometry and AFM

The amount of the protein and the distribution within the spots

were investigated using ellipsometry and AFM. First, the

thickness of the PEO-like coating and the two-drops spots of

fibronectin at different concentrations ranging from 21 up to

333 mg/ml were measured in dry conditions by ellipsometry. The

measurement of the thickness of the PEO-like film between

proteins spots gave a value of 11.5 nm.

Secondly, D and J maps of the protein spots after rinsing

were acquired from which thickness maps were calculated. The

resulting thickness map for the 167, 84 and 42 mg/ml spots is

presented in Fig. 3A, where an increasing spot/background

contrast is visible as a function of the protein concentration. The

effective thickness of the protein layer within the spots was

found to be between 0.15 nm and 0.95 nm for the 42 mg/ml and

333 mg/ml protein concentrations, respectively. The thickness

corresponding to the lowest fibronectin concentration (21 mg/ml)

was not measurable since the values were below the instrument

sensitivity (< 0.1 nm). Since ellipsometry essentially provides the

equivalent thickness of a fully dense organic layer and fibronectin

is a large protein with a length of 15.5 nm and a width of

8.8 nm,33 these calculated thickness values suggest that the

proteins remaining adsorbed on the substrate after rinsing do not

form a fully closed, dense monolayer. The fibronectin mass

densities per area (m) were calculated from the effective thick-

nesses using eqn 1. The results are reported in Table 1 and

presented in logarithmic scale in Fig. 3B as a function of the

initial concentration of the spotted protein solutions, including

their standard deviations, which are based on the measurement

by ellipsometry of six different spots for each concentration. The

calculated values of the protein mass densities for the presented

set of measurements range between 18 ng/cm2 and 113 ng/cm2 for

the 42 mg/ml and 333 mg/ml concentrations, respectively. The

data points could best be fitted with a sigmoidal curve of the form

m ¼ mmax

cn

k n þ cn, with n ¼ 3.1 and k ¼ 70.9 mg/ml (curve in the

plot of Fig. 3B) to find the maximum coverage. The asymptotic

value for high initial protein concentrations, mmax, is 112 !

4 ng/cm2.

Other samples with the same spot arrays obtained from

different protein concentrations were tested and showed repro-

ducibly the same saturation trend.

The reported mass densities suggest that the protein density in

the spot obtained after rinsing does not increase linearly with the

protein concentration. The fibronectin density increases

pronouncedly with the concentration up to 84 mg/ml and forms

a plateau for higher concentrations. These mass density values

were compared to those expected on the spots before rinsing as

calculated from the concentrations of the spotted solutions, the

drop volume (800 pL) and the diameter of the obtained spots

(230 mm). The data are reported in Table 1. After rinsing, the

Fig. 2 ToF-SIMS images of fibronectin [CN(&)]spotted on PEO-like

coated samples as one-drop spot after overnight incubation in water. The

protein was prepared at different concentrations (21, 42 and 84 mg/ml) in

PBS, pH 7.4 (A) and in printing buffer, pH 5 (B). These images confirm

that the proteins remain stably adsorbed on the PEO-like film after

rinsing and therefore can be used as substrate for cell adhesion studies.

Fig. 3 (A) Thickness maps of Fn spots on plasma-deposited PEO-like

film after rinsing in water and calculated from ellipsometric D and J

maps. The thickness is given in nm with respect to the Si surface and

includes the PEO-like layer (11.5 nm). Fibronectin concentration ¼

167 mg/ml (column 1), 84 mg/ml (columns 2–3) and 42 mg/ml (column 4).

Size of the maps: 2000 mm # 1400 mm. (B) Mass density (m) of the

fibronectin spots after rinsing as a function of the protein concentration

in the spotted solution. It was averaged over six equivalent spots

deposited from a solution with equal protein concentration.


spots show a protein density that is always much lower than the

one before rinsing, which suggests that a large part of the spotted

proteins is not strongly bound on the surface and is removed

after rinsing. For instance, only 22% of the proteins from the

42 mg/ml solution and 17% of the 333 mg/ml solution spotted on

the PEO-like film are retained on the surface. Furthermore, it

was estimated from the protein dimensions that a complete,

densely packed monolayer of Fn would result in a mass density

of 600 ng/cm2, assuming that the molecules are oriented with

their long axis parallel to the surface. This results in low protein

coverage as also reported in Table 1 in % of a monolayer. For the

highest analyzed concentration (333 mg/ml) a coverage of 19% is

reached whereas for the lowest concentration (42 mg/ml)

a coverage is of 3% is obtained. These results confirm that

a considerable part of the protein spotted on the PEO-like film is

weakly bound to the surface and suggests that only a non-

compact protein layer remains on the surface. The latter

conclusion is supported by the AFM results, which confirm the

formation of a not fully packed protein layer for protein solu-

tions up to 333 mg/ml. The AFM results for spots generated from

fibronectin solutions of 42 and 167 mg/ml, after the sample was

rinsed and dried, are presented in Fig. 4. The AFM analysis was

performed in the internal part of the spots. At low protein

concentration, fibronectin (white spots) forms small clusters

which are uniformly distributed on the PEO-like coating

substrate (dark background) as evident in the 5 # 5 mm2

topography image (Fig. 4A). For concentrations higher than

84 mg/ml, the proteins are denser and cover a larger part of the

substrate (Fig. 4C). It is difficult to calculate the protein surface

coverage from the AFM measurements due to the tip convolu-

tion effect which overestimates the results. However, the AFM

data confirm that the protein layer is not fully packed on the

surface even for the highest concentrations tested and that

the topographies within spots derived from protein solutions at

different concentrations are notably different.

The height profiles along the scanning lines of the two

topography images are shown in Fig. 4B and 4D. The peaks in

surface height indicate the presence of fibronectin molecules on

the PEO surface. These peaks reach a maximum height of 8 nm

and their number increases with the protein concentration in the

spotting solution. Their broad height distribution suggests that

the proteins are randomly oriented on the surface. Furthermore,

the fact that the observed peak heights are lower than the protein

dimensions can be due to the dry conditions in which these

samples were analyzed and stored.

These results are in agreement with ellipsometry and AFM

data recently reported to describe the morphology of surfaces

developed for bio- and immonological biosensors. Similarly to

the present study, an incomplete layer of proteins (P.96 pertactin

proteins) formed by immersion on a thiolated surface has been

observed.34

3.3. Protein spot reactivity analyzed by SPRi

Once the quantity and the distribution of the spotted proteins on

the PEO-like film had been evaluated, the biological activity of

the spotted fibronectin molecules was investigated. The active

site of the fibronectin is the RGD sequence which is recognized

by the integrins on the cellular membrane. These ligand–integrin

interactions propagate within the cells and influence cell migra-

tion, tissue organization, cell growth and fate.35 Due to the

dimeric structure of the fibronectin, two binding sequences are

present on each molecule. The availability of these active sites is

probed here a posteriori by performing the recognition process in

which they are involved. Thus, the immunorecognition fibro-

nectin/mouse anti-fibronectin monoclonal antibody RGD

specific (Ab-RGD) was monitored by SPR imaging (Fig. 5). For

this purpose, fibronectin was spotted at different concentrations

as two-drop spots on the SPR slides coated by a PEO-like film,

rinsed in 10 mM PBS and exposed to the flows of 200 mg/ml BSA,

20 mg/ml anti-ovalbumin (Ab-OVA) and 10 mg/ml Ab-RGD as

described in the Experimental Section. The changes in reflectivity

after the flow of BSA, Ab-OVA and Ab-RGD were monitored

and analyzed. The results are reported in Fig. 5A. After BSA was

passed over the spotted slide, a marginal change in reflectivity

was measured for all protein spots and the PEO-like film

(Fig. 5A, left). This demonstrates the strong protein repellent

character of the PEO-like film which is not protein covered in

aqueous solution. The graph also clearly shows the specificity of

the immunorecognition test, since, while the Ab-OVA interacts

very slightly with the fibronectin spots and the PEO-like back-

ground, the interaction of the Ab-RGD with the fibronectin

spots is evident (up to 0.7% reflectivity difference) and also

depends on the protein concentration. In particular, the SPR

signal change relative to this interaction is very low for the lowest

fibronectin concentration tested (21 mg/ml) and the PEO-like film

and reaches a saturation value for the two highest protein

concentrations considered. This indicates that the number of

fibronectin molecules recognized by the Ab-RGD is similar

and maximized for these high concentrations. Thus, both

Table 1 Fibronectin concentrations in the initial spotting solutions, on the spot before and after rinsing and protein spot coverage

Initial proteinconcentration (mg/ml)

Average protein density(ng/cm2) before rinsinga

Average protein density(ng/cm2) after rinsingb Protein coveragec (%)

Protein density standarddeviation (ng/cm2) after rinsingd

333 650 113 19 18.3167 326 101 17 15.484 170 72 12 14.342 82 18 3 4.121 41 — — —

a Calculated from initial protein concentration, 2-drop volume (0.8 nL) and spot area (0.041 mm2). b Calculated from the effective thickness of theprotein layer measured by ellipsometry. c Calculated dividing (b) by the protein density estimated necessary to fully pack the spot (600 ng/cm2). Thisdensity was calculated knowing the dimension of the fibronectin (15.5 and 8.8 nm), its mass (440 kDa) and the spot area. d Calculated frommeasuring six different spots for each concentration by ellipsometry.


Fig. 4 AFM topography images of fibronectin molecules within spots deposited on PEO substrate from 42 mg/ml (A) and 167 mg/ml (C) fibronectin

solutions. They indicate the uniform and concentration dependent distribution of the proteins within the spot. Their height profiles along the highlighted

scanning lines (B–D) suggest protein dimensions below 8 nm and random orientation of the proteins on the surface. The sample was incubated in PBS

for 1 h and rinsed in water prior to AFM measurements.

Fig. 5 (A) Reflectivity change due to the injection of 200 mg/ml BSA, 20 mg/ml anti-ovalbumin (Ab-OVA) and 10 mg/ml mouse anti-fibronectin

monoclonal antibody RGD-specific (Ab-RGD) on spots of different fibronectin concentrations. (B) SPR difference image of the fibronectin array after

the interaction with the 10 mg/ml Ab-RGD.


ellipsometry and SPR imaging indicate the presence of a similar

number of fibronectin molecules on the surface after spotting

from the 167 and 333 mg/ml solutions. The same conclusion can

be drawn by observing the SPR difference image of the fibro-

nectin array after the interaction with the Ab-RGD (Fig. 5B).

Here, the brightness of the spots increases every two rows as

a function of the spotted protein concentration. These results

confirm that fibronectin which is spotted and immobilized on the

PEO-like film exposes the RGD sequence and that therefore

this sequence is available for the integrin mediated cell-surface

interactions. In particular, the SPR imaging data also provide

information on the concentration of Ab-RGD on the fibronectin

spots. From these data it was possible to calculate the number of

Ab that interact with the fibronectin spots as described in the

Experimental section and presented in Table 2. By considering

the number of binding sites of the fibronectin molecules on the

spots as measured by ellipsometry and the molecules of reacted

Ab-RGD, the efficiency of the immunoreaction was calculated

(Table 2). This efficiency is around 12% and remains similar for

fibronectin concentrations equal or higher than 84 mg/ml. For the

42 mg/ml concentration an efficiency of 22% is obtained. This

result suggests that antibodies can recognize antigens present on

the surface at low density with higher efficiency than antigens

that are densely packed on the surface. This effect could be due to

a difference in the packing or orientation of the fibronectin

molecules which occurs during the initial adsorption onto the

PEO-like layer from a low concentrated protein solution.

Table 2 Fibronectin molecules in the initial spotting solutions and on the spot after rinsing. Number of Ab-RGD recognized on the spots and efficiencyof the immunorecognition between fibronectin and Ab-RGD

Initial number of fibronectinmolecules in 2 drops (109)a

Number of fibronectinmolecules/mm2 after rinsingb

Number of Ab-RGDreacted/mm2 on the spotsc Recognition efficiencyd (%)

0.36 1550 350 110.18 1400 360 130.09 1000 240 120.045 250 110 220.023 — 27 —

a Calculated from the initial protein concentration (mg/ml), the 2-drop volume (0.8 nl) and the mass of the single fibronectin molecule (0.73 # 10&9 ng).b Calculated from the effective thickness of the protein layer measured by ellipsometry and the mass of the single fibronectin molecule. c Calculated fromthe antibody density on the fibronectin spots measured by SPR imaging and the mass of the single Ab-RGD (0.25 # 10&9 ng). d Calculated by dividingthe number of Ab by the number of active sites of fibronectin available (number of fibronectin # 2).

Fig. 6 (A) A HUCB-NSCs array on a 10 # 10 fibronectin array

generated on PEO-coated slides after 1 day of cell culture. The spots were

obtained by dispensing different number of drops (from left: 1, 2, 3, 4, 5

drops, two columns for each drop number) and fibronectin concentra-

tions (from top: 10, 21, 42, 84, 167 mg/ml, 2 rows for each concentration).

(B–C) Details of HUCB-NSCs and cell nuclei stained with Hoechst on

a 4-drop, 84 mg/ml fibronectin spot. Fifty thousands of cells/cm2 were

cultured over the spotted sample and maintained in standard culture

conditions in presence of 2% serum. The scale bar is 100 mm.

Fig. 7 (A) HUCB-NSCs density (cell number/mm2) on fibronectin spots

of different sizes and concentrations. The error bar comes from cell

counting from different experiments. (B) Cell number as function of

initial protein solution concentration and cell plating densities (5 # 104

and 104 cells/cm2).


3.4. HUCB-NSCs array

The capability of the protein arrays to address the influence of

different protein types and concentrations on cell behaviour (cell

adhesion, spreading, proliferation, etc.) was verified by culturing

HUCB-NSCs on protein arrays as described in the Experimental

section. Here, we specifically report the effect of fibronectin and

other ECM proteins on cell adhesion.

HUCB-NSCs were seeded on spots having different fibronectin

concentrations and sizes. The cell array resulting after 1 day of cell

culture is shown in Fig. 6A (one 10# 10 spot array). The cell array

is well defined and the effects of spot size and protein concentra-

tion on cell adhesion are visible. In particular, optical images of

HUCB-NSCs on a 4-drops, 84 mg/ml fibronectin spot before and

after nuclei staining are presented in Fig. 6 B–C.

The data obtained from the analysis of spots from three

different experiments are reported in Fig. 7A. Here, cells were

cultured the day after substrate preparation and fixed after 1 day

of culture. The histogram in Fig. 7A shows that the cell density

depends on the initial protein concentration (21 and 84 mg/ml are

presented) and not on the spot size. The large error bars (typi-

cally <23%) derive from protein and cell solutions variability.

Fig. 7B represents the number of cells present on the fibronectin

spots (4-drop spot) as function of the initial protein concentra-

tion. It reveals clearly that for low plating densities (1 # 104 cells/

cm2) the number of cells increases slowly with the protein

concentration. For high cell density (5 # 104 cells/cm2) the cell

number increases sensibly at low protein concentration and

stabilizes at around 135 cells for concentration above 42 mg/ml.

We can notice from these results first that cells recognize protein

concentrations (10 and 21 mg/ml) which the refractivity methods

used in this study are not able to characterize due to sensitivity

limits. Secondly, the cells can adhere on the spots when the

density of the fibronectin molecules, relative to the initial protein

concentration of 42 mg/ml, is 18 ng/cm2 and the protein coverage

is only 3%. This means that the protein distribution as presented

in Fig. 4A as topography image is high enough to allow cell

adhesion and the formation over time of a cell monolayer on the

spotted area under our experimental conditions. Cell adhesion

and growth on low dense nanofabricated surfaces has been

systematically studied elsewhere.36

To assess if the protein buffer can alter the anti-adhesive

property of the PEO-like film, the cells were also cultured on

spots on which only printing buffer was spotted. No cells were

found on these spots confirming that cell adhesion is only due to

the presence of the proteins.

Fig. 8 HUCB-NSCs on fibronectin (A), laminin (B) and collagen V (C) after 4 days of culture on 3-drop protein spots. The effect of different protein

types and protein concentrations are visible. Fifty thousands of cells were seeded and maintained in standard culture conditions in presence of 2% serum.

The scale bar is 100 mm.


HUCB-NSCs were culture also on other proteins, including

collagen I, collagen III and collagen V, laminin, BSA and the

polypeptide poly-L-lysine (PLL). The different affinity of the

cells to the tested substrates was confirmed by contrast phase

observations (data not shown). For the collagens, the order of

affinity based on the number of adherent cells counted on the

respective spots was: collagen III < collagen I < collagen V. Cells

do not adhere on BSA (which can be used as negative control),

while remaining rounded but actively attached to PLL spots.

HUCB-NSCs on fibronectin, laminin and collagen V spots at

three different concentrations are presented in Fig. 8. Note also

that the three drops of the different proteins give the same spot

size of about 260 mm (3-drop spot) on the PEO-like coating. It is

evident that the shape and number of the cells on the different

protein spots are protein type and concentration dependent. The

cells adhere well on fibronectin and cover the entire spot area of

the two concentrations considered included the lower concen-

tration presented (Fig. 8A). On laminin, cells have a different

behavior since they do not spread out but attach only to the spot

edges for low protein concentration spot (21 mg/ml) and cover the

entire spot area for higher concentration (84 mg/ml) always

maintaining a rounded shape (Fig. 8B). HUCB-NSCs adhere

well also on collagen V even if cells are less numerous as

compared to those that contain fibronectin. For the three

collagen V concentrations, an increase of the number of cells

attached to the spots is visible (Fig. 8C). Furthermore, for

laminin and collagen V the tendency of the cells to attach first to

the boundary of the protein patterns is also evident.16

This different cell adhesion behavior agrees well with the

expression profile of HUCB-NSCs. Neurally committed cells, as

compared to the starting population of cord blood mononuclear

cells, revealed up-regulation of Collagen V, Collagen III and

Collagen IX. In addition they express b1, a1 and a3 integrin

subunits which in combination form fibronectin and laminin

(b1 with a3) and collagen V (b1 with a1) receptors.37,38

The differentiation of HUCB-NSCs can be spontaneous

(induced by cell adhesion) or induced by soluble factors

(including serum).38 HUCB-NSCs growing for 1 week on

fibronectin spots in the presence of 2% serum revealed positive

staining specific for b tubulin III and S-100b, showing coexis-

tence of cells differentiated into neuronal and astroglial cells on

the same spot (Fig. 9). Other experiments showed that HUCB-

NSCs can be cultured in serum-free conditions on fibronectin

spots with concentrations higher than 84 mg/ml. For lower

concentrations, the cells detached from the protein spots after

few days of culture. The possibility of controlling the adhesive

surfaces and working in serum free-conditions will allow a better

understanding of the role that single ECM proteins and/or

mixtures of them and soluble factors play separately or in

combination on cell differentiation. Successful culture of

embryonic and somatic stem cells in serum deprived conditions,

free from animal product, have been recently reported.39,40 Such

approaches allow controlled drug testing and possible direct

clinical application of stem cells.

4. Conclusions and outlook

We have presented a novel method for patterning fibronectin for

creating well defined stem cell microarrays. The technique is

based on the direct spotting of proteins on a plasma polymerized

poly(ethyleneoxide) film which has amphipathic properties

i.e. protein repellent in wet conditions and protein adhesive when

proteins are spotted or micro-contact printed.

A qualitative and quantitative analysis of fibronectin spots

generated by this technique show that it is possible to create

a stable layer of proteins with a controlled density. The good

activity of the spotted fibronectin molecules has been demon-

strated by monitoring their interactions with antibodies that are

specific to cell binding sites. HUCB-NSCs were seeded on

fibronectin spots having different concentrations and sizes. Well

defined cell microarrays have been generated with a good

stability, which did not show cell migration for several days. The

cell array properties, i.e., cell densities versus spot size and

protein concentrations, have been quantified. The results show

that the cells adhere and cover the fibronectin spots well, even at

low protein concentrations. Different adhesion is reported for

other kind of ECM proteins, BSA and PLL.

These results show that the PEO-like film is an interesting

alternative platform thanks to the high stability of its anti-

adhesive properties during extended cell culturing. Protein

array platforms developed in this study and the optimized cell

culture protocol provide a promising approach to investigate

also in a high throughput manner how insoluble factors

patterned on the surface influence stem cell adhesion and

development. The investigation of the long term effects of

mixtures of proteins or protein layers and gradients on cell

differentiation is foreseen.

Acknowledgements

This work has been supported by the funding for the Exploratory

Research Projects 2007 by the IHCP and continued in the frame

of the Institutional Project 15008 ‘‘NanoBiotechnology for

Health’’, Institutional Project 15003 ‘Validation for chemicals

and correlate’ and Institutional project 15005 ‘Validation for

consumer products’. The authors want to thank E. Parnisari for

the PEO-like coating deposition.

Fig. 9 HUCB-NSCs on a fibronectin spot stained with monoclonal

antibodies against neuronal b tubulin 3 (TUJ1, green) and polyclonal

rabbit antibodies against S-100b (red) specific for neuronal and astroglial

cells, respectively. In blue the cell nuclei stained by Hoechst are visible.

The cell array to which the spot belongs to was fixed and stained after 1

week of cell culture. The scale bar is 100 mm.


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Fabrication and characterization of protein arrays for stem cell patterning

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