Fabrication of a cell array on ultrathin hydrophilic polymer gels utilising electron beam irradiation and UV excimer laser ablation.

Most of the surface patterning methods currently applied are based on lithography techniques and microfabrication onto silicon or glass substrates. Here we report a novel method to prepare patterned surfaces on polystyrene substrates by grafting ultrathin cell-repellent polymer layers utilising both electron beam (EB) polymerisation and local laser ablation techniques for microfabrication. Polyacrylamide was grafted onto tissue culture polystyrene (TCPS) dishes using EB irradiation. Water contact angles for these PAAm-grafted TCPS surfaces were less than 10 degrees (costheta = 0.99) with PAAm grafted amounts of 1.6 microg/cm(2) as determined by ATR/FT-IR. UV excimer laser (ArF: 193 nm) ablation resulted in the successful fabrication of micropatterned surfaces composed of hydrophilic PAAm and hydrophobic basal polystyrene layers. Bovine carotid artery endothelial cells adhered only to the ablated domains after pretreatment of the patterned surfaces with 15 microg/mL fibronectin at 37 degrees C. The ablated domain sizes significantly influenced the number of cells occupying each domain. Cell patterning functionality of the patterned surfaces was maintained for more than 2 months without loss of pattern fidelity, indicating that more durable cell arrays can be obtained compared to those prepared by self-assembled monolayers of alkanethiols, as described in previous reports. The surface fabrication techniques presented here can be utilised for the preparation of cell-based biosensors as well as tissue engineering constructs.

[1]  A. Shard,et al.  Cellular attachment and spatial control of cells using micro-patterned ultra-violet/ozone treatment in serum enriched media. , 2004, Biomaterials.

[2]  M C Davies,et al.  Spatially controlled cell engineering on biodegradable polymer surfaces , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[3]  K. Healy,et al.  Protein adsorption and cell attachment to patterned surfaces. , 2000, Journal of biomedical materials research.

[4]  T. Okano,et al.  A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). , 1993, Journal of biomedical materials research.

[5]  George M Whitesides,et al.  Propagation of viruses on micropatterned host cells. , 2003, Biotechnology and bioengineering.

[6]  T. Okano,et al.  Thermo‐responsive polymeric surfaces; control of attachment and detachment of cultured cells , 1990 .

[7]  M. Toner,et al.  Cellular Micropatterns on Biocompatible Materials , 1998, Biotechnology progress.

[8]  G. Whitesides,et al.  Patterning Mammalian Cells Using Elastomeric Membranes , 2000 .

[9]  F. Grinnell,et al.  Fibronectin adsorption on hydrophilic and hydrophobic surfaces detected by antibody binding and analyzed during cell adhesion in serum-containing medium. , 1982, The Journal of biological chemistry.

[10]  Gaudenz Danuser,et al.  Microcontact printing of novel co-polymers in combination with proteins for cell-biological applications. , 2003, Biomaterials.

[11]  F. Grinnell Fibronectin Adsorption on Material Surfaces a , 1987, Annals of the New York Academy of Sciences.

[12]  C. Chan,et al.  Polymer surface modification by plasmas and photons , 1996 .

[13]  B. Dalton,et al.  Effects of polystyrene surface chemistry on the biological activity of solid phase fibronectin and vitronectin, analysed with monoclonal antibodies. , 1993, Journal of cell science.

[14]  C. S. Chen,et al.  Geometric control of cell life and death. , 1997, Science.

[15]  Y. Ito,et al.  Surface micropatterning to regulate cell functions. , 1999, Biomaterials.

[16]  Shunsuke Koike,et al.  Nanofabrication for micropatterned cell arrays by combining electron beam-irradiated polymer grafting and localized laser ablation. , 2003, Journal of biomedical materials research. Part A.

[17]  Mitsuo Umezu,et al.  The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets. , 2005, Biomaterials.

[18]  T. Okano,et al.  Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface. , 2000, Biomacromolecules.

[19]  Terri Adams,et al.  Streamlining the Drug Discovery Process by Integrating Miniaturization, High Throughput Screening, High Content Screening, and Automation on the CellChip™ System , 1999 .

[20]  T. Okano,et al.  Novel patterned cell coculture utilizing thermally responsive grafted polymer surfaces. , 2001, Journal of biomedical materials research.

[21]  M L Yarmush,et al.  Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3T3 fibroblasts. , 1997, Journal of biomedical materials research.

[22]  J. Lhoest,et al.  Fibronectin-pluronic coadsorption on a polystyrene surface with increasing hydrophobicity: relationship to cell adhesion. , 1999, Journal of biomedical materials research.

[23]  Daniel I. C. Wang,et al.  Engineering cell shape and function. , 1994, Science.

[24]  Teruo Okano,et al.  [Cell sheet engineering]. , 2004, Rinsho shinkeigaku = Clinical neurology.

[25]  D J Beebe,et al.  Microfabricated elastomeric stencils for micropatterning cell cultures. , 2000, Journal of biomedical materials research.

[26]  Joseph D. Andrade,et al.  The Contact Angle and Interface Energetics , 1985 .

[27]  T. Okano,et al.  Two-dimensional manipulation of confluently cultured vascular endothelial cells using temperature-responsive poly(N-isopropylacrylamide)-grafted surfaces. , 1998, Journal of biomaterials science. Polymer edition.

[28]  Masayuki Yamato,et al.  Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture. , 2002, Biomaterials.

[29]  Masayuki Yamato,et al.  Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[30]  D E Ingber,et al.  Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver. , 1997, Experimental cell research.

[31]  J. Steele,et al.  Role of serum vitronectin and fibronectin in adhesion of fibroblasts following seeding onto tissue culture polystyrene. , 1992, Journal of biomedical materials research.

[32]  H. Busscher,et al.  Plasma-treated polystyrene surfaces: model surfaces for studying cell-biomaterial interactions. , 2004, Biomaterials.

[33]  Yoshito Ikada,et al.  Effect of preadsorbed proteins on cell adhesion to polymer surfaces , 1993 .

[34]  D. Boettiger,et al.  Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation. , 1999, Molecular biology of the cell.

[35]  Milan Mrksich,et al.  Self-assembled monolayers of alkanethiolates presenting mannitol groups are inert to protein adsorption and cell attachment , 2000 .