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Applied Surface Science 257 (2011) 5250–5254

Contents lists available at ScienceDirect

Applied Surface Science

journa l homepage: www.e lsev ier .com/ locate /apsusc

D spatially controlled polymer micro patterning for cellular behavior studies

. Dincaa,∗, A. Palla-Papavlua, I. Paraicob, T. Lippertc, A. Wokaunc, M. Dinescua

NILPRP, National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, Zip RO-077125, Magurele, Bucharest, RomaniaDepartment of Cellular and Molecular Medicine, “Carol Davila” Medical University, Eroii Sanitari No 8, Bucharest, RomaniaPaul Scherrer Institute, General Energy Research Department, 5232 Villigen PSI, Switzerland

r t i c l e i n f o

rticle history:

a b s t r a c t

vailable online 7 October 2010

eywords:olyethyleneimineolyethyleneglycol

A simple and effective method to functionalize glass surfaces that enable polymer micropatterning andsubsequent spatially controlled adhesion of cells is reported in this paper. The method involves theapplication of laser induced forward transfer (LIFT) to achieve polymer patterning in a single step ontocell repellent substrates (i.e. polyethyleneglycol (PEG)). This approach was used to produce micron-sizepolyethyleneimine (PEI)-patterns alternating with cell-repellent areas. The focus of this work is the abilityof SH-SY5Y human neuroblastoma cells to orient, migrate, and produce organized cellular arrangements

ttern

atternseuronal cells

on laser generated PEI pa

. Introduction

Manipulating and guiding the cellular adhesion and migrationor the formation of various cellular networks in vitro is of highnterest both in medical basic research and for biotechnologicalpplications.

By defining the design and the direction of cellular outgrowthnd connectivity on artificial surfaces it offers potential applicationn the design of prostheses and implants (i.e. which contain graftedells) or in the creation of novel biosensors or microfluidic devices.his selective surface modification at the micrometer-scale repre-ents the necessary step to direct cellular adhesion and growth intoatterns, implying various types of modifications, from chemicalurface modification [1,2] to topographical surface modifications3,4] or combinations of both [5].

Material characteristics are an important issue, especially whenhey are used in special environments. It is known that cell adhesiveoatings do not maintain their surface properties in a physiologicalnvironment. One approach to enhance cell adherence on a mate-ial that should act as cell adherent coating is the utilization ofhe possible electrostatic interaction between positively chargedmino-groups and negatively charged phospholipids in the cellembrane. Collagen, fibronectin, laminin, or synthetic polymeric

ations, such as polylysine or polyornithine, have also been used

s attachment promoting factors in numerous studies [6–9]. How-ver, one of the main drawback in the use of the most polymerss the presence of peptide-like amide linkages onto a syntheticolymer backbone which would imply dehydration process and

∗ Corresponding author. Tel.: +40 2145744142; fax: +40 2145744142.E-mail address: dinali@nipne.ro (V. Dinca).

169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.apsusc.2010.09.113

s.© 2010 Elsevier B.V. All rights reserved.

a lost in the structural, mechanical and biological properties ofthe polymer [8–11]. An interesting alternative is polyethylenimine(PEI) which can avoid the presence of this type of linkage in thepolymer backbone. Its electrostatic properties and moreover thechemical stability promotes a stable binding between cell mem-branes and negatively charged surfaces (e.g. mica, silicon dioxide)[12–14].

Methods such as microlithography or micro-contact printinghave been used as tools to produce topographical and chemicalpatterns. The main drawback of these methods is related to con-taminations, a limited number of materials that can be used forobtaining patterns, and the application of masks and stamps. Dur-ing the last years, lasers proved to be useful tools for fast andaccurate patterning of various sensitive materials [15,16], suchas DNA [17], proteins [18–21], cells [22], or synthetic polymers[23,24]. In conventional laser-induced forward transfer (LIFT), atransparent substrate is coated with the material to be transferred,i.e. the donor, and placed close to a receiver substrate. The laserbeam passes through the donor substrate on a precise spot of thefilm and results in its deposition on the receiver. In order to avoidany damage of the sensitive transfer material by the laser irradi-ation, a sacrificial layer or dynamic release layer (DRL) can be used[25,26]. The aim of this paper is to obtain two dimensional spatiallycontrolled PEI micro patterns using DRL assisted LIFT and to studythe adhesion and cellular behavior of SH-SY5Y human neuroblas-toma cells on these patterns.

2. Materials and methods

The transfer was achieved using a single pulse from a XeClexcimer laser (Compex, Lambda Physik, 308 nm, 30 ns). A squaremask with variable aperture was applied to utilize a homogeneous

V. Dinca et al. / Applied Surface Science 257 (2011) 5250–5254 5251

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ig. 1. SEM images of PEI pixels obtained with the high laser fluence of 520 mJ cm−2

art of the beam, which was focused by a lens onto the backsidef the donor film giving a spot size of 200–300 �m. The donor andhe receiver substrates were placed in contact perpendicular to theeam, on a motorized translation stage.

The computer-controlled system allows creating a matrix ofixels for each sample, where the pulse energy and the distanceetween the PEI pixels (from 50 to 250 �m) are varied. Images wereaken by an optical microscope (Zeiss Axioplan) coupled with a dig-tal camera (Leica DC500) and by a Scanning Electron MicroscopeSEM).

All transfer experiments were performed in air. To avoid directaser damaging, an additional triazene polymer (TP) layer with atrong absorption at the applied laser wavelength of 308 nm, was

pplied as dynamic release layer [25–27].

The multilayer donor films were prepared by coating succes-ively fused silica substrates with the TP and the materials toransfer (PEI). Films of the TP were prepared by spin coating fromolutions in chlorobenzene and cyclohexanone (1:1, w/w) with

d with a laser fluence below 400 mJ cm−2 (b). The scale bar corresponds to 200 �m.

final thicknesses of around 150 nm. PEI, 1.5% in ethanol was spin-coated on top of the triazene layer and thin films with thicknessesof about 100 nm were obtained.

The exact thickness of the films was determined by a surfaceprofiler (Dektak 8000). The receiver substrates were glass platescoated with polyethyleneglycol (PEG) polymer. The coating proto-col was the same as for the TP, and the used solution was 2% PEGin water.

Optical Microscopy images were used to analyze the morphol-ogy of the deposited pixels and of the adhesion of neural cells onthe patterns.

To obtain the cell cultures, the receiver surfaces were washedgently twice with sterile SF-HBSS (Serum-free Hank’s balanced

salt solution), and placed in 6-cm Petri dishes. 5 ml of SH-SY5Y human neuroblastoma cell suspension in DMEM (Dulbecco’smodified Eagle’s medium) with phenol red, 10% FCS and 0.1% peni-cillin/streptomycin, were placed over the surface of the cells andkept in a CO2 incubator at 37 ◦C.

5252 V. Dinca et al. / Applied Surface Science 257 (2011) 5250–5254

Fig. 2. Phase contrast images of (a) PEI array in culture medium, (b) SH-SY5Y distribution on patterned surfaces after three days in vitro and (c) an inset of SH-SY5Y distributiono

Fp(

n a PEI pixel after three days in vitro.

ig. 3. Phase contrast images of a PEI array in culture medium (a) the PEI pixels are arrarinted on the PEG coated support at distances below 50 �m. (c and d) SH-SY5Y neural cee) An inset of a region of a PEI pixel and the surrounding PEG coated area cultured with S

nged in the array at distances of approximately 200 �m and (b) the PEI pixels arells after 3 days in vitro attached on the PEI pixels transferred at different distances.H-SY5Y neuronal cells after one day in vitro.

ce Science 257 (2011) 5250–5254 5253

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V. Dinca et al. / Applied Surfa

. Results and discussions

The patterns that can be obtained by LIFT are strongly influencedy the transfer process parameters. In a previous study [27] weould show that only a narrow operating window can be applied forbtaining polymer patterns with well-defined shape, in a preciseocation and in a controlled manner. The key parameters are relatedo a minimal thickness of the dynamic release layer required toenefit fully of its advantages, the laser fluence, the thickness ratiof PEI to the TP, and the transfer distance [27]. For all the samples,he fluence at which a complete detachment of the polymer pixelccurs was determined by visual inspection of the donor substrates.

For a donor with a TP layer thickness of 150 nm and PEI layerf 100 nm, transfer without any type of debris or splashes aroundhe pixels was achieved for rather high fluences of ∼520 mJ cm−2

Fig. 1a). By decreasing the laser fluence (below 400 mJ cm−2), theixels keep a regular shape, but with a different surface aspect andith missing parts inside the pixel (Fig. 1b). This corresponds to

ur previous observation, where clean transfer was only achievedith a TP layer thicker than the PEI layer and for a similar range of

aser fluences.However, the focus of this work is the ability of SH-SY5Y human

euroblastoma cells to orient, migrate, and produce organized cel-ular arrangements on PEI patterns. Thus, only the pixels whichxhibit a well-defined shape, are complete, and debris free will besed for cell culturing experiments.

Fig. 2 illustrates the evolution of the cell adhesion on the PEI-attern. The PEI array on the PEG coated surface in the cultureedium is shown in Fig. 2a. In Fig. 2b is shown the SH-SY5Y human

euroblastoma cell suspension in vitro on the receiver substratesfter three days. In Fig. 2c is shown an inset SH-SY5Y neuronal cellsistributed inside of a PEI pixel. The clustering of neurons inside theEI pixel is clearly visible, while outside the pixel area randomlyistributed neurons may be found (Fig. 2c).

The results emphasize a typical phenomena observed duringhe experiments. Neuronal adhesion is almost negligible on PEGubstrates while for PEI areas an excellent neurophilic coating isbtained, which supports the adhesion of the neurons inside theEI pattern significantly. Statistical analysis revealed that 85 ± 10%f the total number of observed neurons is located inside the PEI-oated patterned surfaces.

Even after 24h of incubation, the neurons manifest a clear affin-ty to the PEI pixel surface (visible in Fig. 3e), this representing therst step for the neurons to arrange into clusters inside the poly-er patterns. Outside the PEI polymer pixel the neurons are only

echanically attached and they can be removed from the surface

y gentle washing with culture medium.The results of cell aggregation on PEI-patterns obtained by

IFT and for PEI-coated glass which has been prepared by spinoating are comparable. The adhesion of separate neurons onto

Fig. 5. PEG coated glass substrates in culture medium

Fig. 4. Average number of observed interconnecting neurite fascicles vs. the sep-aration distance between the transferred PEI-pixels. The mean plus the standarddeviation (calculated over 5 images) is included.

the glass was not taken into account because individual neu-rons cannot be identified due to the cell aggregation. A fluenceof 520 mJ cm−2 was used to obtain polymer patterns with varyingdistances between PEI pixels. In this study distances in the range50–250 �m were investigated. In Fig. 3a and b are shown PEI pixelsprinted onto PEG coated substrates at extreme values, respectively50 �m and 200 �m. Furthermore, in Fig. 3c and d are presented theinterconnecting neurite fascicles between PEI-patterns with sep-aration distances between the PEI-pixels of 50 �m and 150 �mrespectively. The center of neuron aggregates is frequently posi-tioned inside the PEI-pattern if the PEI-patterns separated bylarger distances (see Fig. 3c). On patterns with pixels separatedby small distances, cellular aggregates are also found in betweenthe pixels, but they form usually interconnecting neurite fascicles(see Fig. 3d).

An inverse proportional relation between the number of inter-connecting neurite fascicles and the separation distance betweenthe pixels after 3 days in vitro is obtained. The minimal distanceshould be used, because for patterns with a separation distance ofhundreds of microns, no interconnecting neurite fascicles betweenPEI-coated pixels are observed. The development of fasciculatedneurites for neurons on the PEI-pattern is faster and increase innumber, i.e. with an average of 6 neurites, while the neurons

located outside the PEI-pattern develop an average of 3 neurites.The dependence of the observed interconnecting neurite fascicleson the separation distance between the PEI-pixels is shown in Fig. 4.As stated above, the maximum distance for which the neuronsdevelop more than 3 neurite fascicles is 50 �m. This reveals that

after (a) 3 days in vitro and (b) 7 days in vitro.

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ot only the size of the patterns affects the cells but also that thebility of the cells to affect the micropatterned substrates plays aole.

In Fig. 5 is shown an area of the surface of a PEG coated glassupport in culture medium after (a) 3 days and (b) 7 days. A degra-ation of the inert or non adhesive cell surface after 3 days ofulture is observed. This is probably due to the possibility that cellsecrete cellular matrix or additional enzymes that could degradend modify the composition of the inert surfaces. This cellularrocess which causes degradation changes the local microenviron-ent but depends on the type of cells being patterned and their

ulture conditions [28].The factors mentioned above, i.e. the distance between the

rinted PEI-pixels and the degradation of the non-adherent cellurface, must be taken into account when designing patterned sur-aces. Therefore, preincubation of these substrates for up to 3 daysn cell media did not appear to significantly affect the stability ofEI pixels, while the patterns on substrates that have been prein-ubated in media for more than 1 week showed exfoliation. Thesebservations suggest that the non-adhesive PEG degrades gradu-lly by a cell-independent mechanism while by adding neural cellshich have active cellular processes; the natural rate of degrada-

ion can even increase.

. Conclusions

After 3 days of observation a general trend is revealed, i.e. thatlarge number of neurons located on the laser transferred PEI-

atterns, forms aggregates after an active migration of the adheringells towards each other. The development of interconnecting neu-ite fascicles is significantly higher for neurons adhered on theaser-transferred PEI-pattern.

The distance between the polymer pixels is a key parametern the orientation and migration of SH-SY5Y human neuroblas-oma cells. A complete isolation of neurons between neurophilicEI-coated patterns on PEG substrates is obtained with a mini-al separation distance of 100 �m which additionally serves asbarrier for the development of a large number of interconnectingeurites.

The observations of exfoliation of the inert coatings after 3 daysn the presence of cell medium can be useful for the design ofell-based biosensors which are able to detect the activation ornhibition of specific enzymes.

cknowledgements

Financial support from the NATO SfP 982671 project and theational Project 71-111 FOTOPOL are gratefully acknowledged. Theuthors would like to thank M. Nagel for synthesizing the triazeneolymer and J. Shaw-Stewart for providing the substrates with apin-coated triazene polymer layer.

eferences

[1] D. Kleinfeld, K.H. Kahler, P.E. Hockberger, Controlled outgrowth of dis-

sociated neurons on patterned substrates, J. Neurosci. 8 (1988) 4098–4120.

[2] J.M. Corey, B.C. Wheeler, G.J. Brewer, Micrometer resolution silane-based pat-terning of hippocampal neurons: critical variables in photoresist and laserablation processes for substrate fabrication, IEEE Trans. Biomed. Eng. 43 (1996)944–955.

[

ence 257 (2011) 5250–5254

[3] P. Clark, P. Connolly, A.S.G. Curtis, J.A.T. Dow, C.D.W. Wilkinson, Topographi-cal control of cell behavior: II. Multiple grooved substrata, Development 108(1990) 635–644.

[4] P. Clark, Cell behavior on micropatterned surfaces, Biosensors Bioelectron. 9(1994) 657–661.

[5] D.V. Nicolau, T. Taguchi, H. Taniguchi, H. Tanigawa, S. Yoshikawa, Patterningneuronal and glia cells on light-assisted functionalised photoresists, BiosensorsBioelectron. 14 (1999) 317–325.

[6] M. Mrksich, C.S. Chen, Y. Xia, L.E. Dike, D.E. Ingber, G.M. Whitesides, Control-ling cell attachment on contoured surfaces with self-assembled monolayers ofalkanethiolates on gold, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 10775–10778.

[7] Singhvi**, A. Kumar, G.P. Lopez, G.N. Stephanopoulos, D.I.C. Wang, G.M. White-sides, D.E. Ingber, Engineering cell shape and function, Science 264 (1994)696–698.

[8] H.K. Kleinman, R.J. Klebe, G.R. Mart, Role of collagenous matrices in the adhesionand growth of cells, J. Cell Biol. 88 (1981) 473–485.

[9] H.K. Kleinman, L. Luckenbill-Edds, F.W. Cannon, G.C. Sephel, Use of extra-cellular matrix components for cell culture, Anal. Biochem. 166 (1987) 1–13.

10] E. Yavin, Z. Yavin, Attachment and culture of dissociated cells from rat embryocerebral hemispheres on polylysine-coated surface, J. Cell Biol. 62 (1974)540–546.

11] P.C. Letourneau, Possible roles for cell-to-substratum adhesion in neuronalmorphogenesis, Dev. Biol. 44 (1975) 77–91.

12] D. Horn, Polyethylenimine-physicochemical properties and applications, in:E.J. Goethals (Ed.), Polymeric Amines and Ammonium Salts, Pergamon, Oxford,UK, 1980, pp. 333–355.

13] I.H. Lelong, V. Petegnief, G. Rebel, Neuronal cells mature faster on polyethylen-imine coated plates than on polylysine coated plates, J. Neurosci. Res. 32 (1992)562–569.

14] T.P. Golub, A.L. Skachkova, M.P. Sidorova, Investigation of surface reactions onan interface of SiO with an aqueous solution of polyethylenimine, Coll. J. Russ.Acad. Sci.** 54 (1991) 697–702.

15] C.B. Arnolsd, P. Serra, A. Pique, Laser direct-write techniques for printing ofcomplex materials, MRS Bull. 32 (2007) 23–31.

16] A. Pique, D.B. Chrisey, J.M. Fitz-Gerald, R.A. McGill, R.C.Y. Auyeung, H.D. Wu,S. Lakeou, V. Nguyen, R. Chung, M. Duignan, Direct writing of electronic andsensor materials using a laser transfer technique, J. Mater. Res. 15 (2000) 1872–1875.

17] P. Serra, M. Colina, J.M. Fernandez-Pradas, L. Sevilla, J.L. Morenza, Preparationof functional DNA microarrays through laser-induced forward transfer, Appl.Phys. Lett. 85 (2004) 1639–1641.

18] I. Zergioti, A. Karaiskou, D.G. Papazoglou, C. Fotakis, M. Kapsetaki, D. Kafet-zopoulos, Femtosecond laser microprinting of biomaterials, Appl. Phys. Lett.86 (2005) 163902–163903.

19] M. Duocastella, J.M. Fernandez-Pradas, J. Dominguez, P. Serra, J.L. Morenza,Printing biological solutions through laser-induced forward transfer, Appl.Phys. A 93 (2008) 941–945.

20] V. Dinca, E. Kasotakis, J. Catherine, A. Mourka, A. Mitraki, A. Popescu, M. Dinescu,M. Farsari, C. Fotakis, Development of peptide-based patterns by laser transfer,Appl. Surf. Sci. 254 (2007) 1160–1163.

21] V. Dinca, A. Ranella, M. Farsari, D. Kafetzopoulos, M. Dinescu, A. Popescu, C.Fotakis, Quantification of the activity of biomolecules in microarrays obtainedby direct laser transfer, Biomed. Microdev. 10 (2008) 719–725.

22] D.B. Chrisey, A. Doraiswamy, R.J. Narayan, T. Lippert, L. Urech, A. Wokaun, M.Nagel, B. Hopp, M. Dinescu, R. Modi, R.C.Y. Auyeung, Two-dimensional dif-ferential adherence of neuroblasts in laser micromachined CAD/CAM agarosechannels, Appl. Surf. Sci. 252 (2006) 4748–4753.

23] V. Dinca, A. Palla Papavlu, A. Matei, C. Luculescu, M. Dinescu, T. Lippert, F. DiPietrantonio, D. Cannata, M. Benetti, E. Verona, A comparative study of DRL-liftand lift on integrated polyisobutylene polymer matrices, Appl. Phys. A. (2010)doi:10.1007/s00339-010-5826-6.

24] C. Boutopoulos, V. Tsouti, D. Goustouridis, S. Chatzandroulis, I.** Zergioti, Liquidphase direct laser printing of polymers for chemical sensing applications, Appl.Phys. Lett. 93 (2008), 191109-1/3**.

25] L. Urech, Thomas Lippert: “Designed polymers for ablation”, in: Laser Abla-tion and its Applications, Springer Series in Optical Sciences, vol. 129, Springer,2007, pp. 281–297.

26] M. Nagel, R. Hany, T. Lippert, M. Molberg, F. Nuesch, D. Rentsch, Aryltriazenephotopolymers for UV-laser applications: improved synthesis and photode-composition study, Macromol. Chem. Phys. 208 (2007) 277– 286.

27] V. Dinca, R. Fardel, F. Di Pietrantonio, D. Cannatà, M. Benetti, E. Verona, A.

Palla-Papavlu, J. Shaw-Stewart, M. Dinescu, T. Lippert, Laser-induced forwardtransfer: an approach to single-step polymer microsensor fabrication, SensorsLett. 8 (2010) 436–440.

28] M. Nelson, S. Raghavan, J.L. Tan, C.S. Chen, Degradation of micropatternedsurfaces by cell-dependent and -independent processes, Langmuir 19 (2003)1439–1449.