Microcontact printing and microspotting as methods for direct protein patterning on plasma deposited polyethylene oxide: application to stem cell patterning Ana Ruiz & Marzena Zychowicz & Laura Ceriotti & Dora Mehn & Lucel Sirghi & Hubert Rauscher & Ilaria Mannelli & Pascal Colpo & Leonora Buzanska & François Rossi Published online: 13 February 2013 # Springer Science+Business Media New York 2013 Abstract Two methods for protein patterning on antifoul- ing surfaces have been applied to analyze the density and bioactivity of the proteins after deposition. Microcontact printing has been used as a technique to transfer fibronectin through conformal contact, while piezoelectric deposition has been employed as a non-contact technique for producing arrays of fibronectin (FN). Plasma deposited polyethylene oxide-like (PEO-like) films have been used as non-fouling background to achieve the bioadhesive/biorepellent surface contrast. Both patterning methods allow the direct fabrica- tion of protein arrays on a non-fouling substrate, and the subsequent formation of a pattern of stem cells by cell attachment on the arrayed substrates. Microcontact printing produced fully packed homogeneous fibronectin patterns, much denser than microspotted patterns. Both printing and spotting technologies generated functional protein arrays, their bioactivity being primarily modulated by the density of the deposited protein layer. Optimization of the FN parameters used for deposition has lead to the achievement of high-quality microarrays with large population of neural stem cells immobilized in the patterns in serum-free con- ditions, where cells exhibit a more homogeneous starting population and factors influencing fate decisions can be more easily tracked. The immunorecognition of fibronectin targeted antibodies, as well as the cell density, increase with the protein density up to a saturation point. Over 100 ng/cm 2 of fibronectin on the surface leads to a decrease in the number of attached cells and a raise of cell spreading. Keywords Micropatterning . Surface modification . Protein . Cell adhesion . Stem cells 1 Introduction Considerable efforts have been recently done to tailor at the microscale the physico-chemical surface properties of bio- materials in order to modulate in-vitro and in-vivo cellular responses required by specific applications. Integrins are the transmembrane receptors responsible for the regulation of such cellular responses to biomaterials. Integrin-mediated cell adhesion involves receptor-ligand binding and post- ligation interactions which lead to the formation of focal adhesion supramolecular complexes and the eventual Electronic supplementary material The online version of this article (doi:10.1007/s10544-013-9749-9) contains supplementary material, which is available to authorized users. A. Ruiz (*) : L. Ceriotti : D. Mehn : L. Sirghi : H. Rauscher : I. Mannelli : P. Colpo : F. Rossi (*) European Commission, Joint Research Centre, Institute for Health and Consumer Protection, TP 203, Via Fermi, 21027 Ispra, VA, Italy e-mail: aruizmo@gmail.com e-mail: francois.rossi@jrc.ec.europa.eu A. Ruiz e-mail: ana.ruiz@unimi.it M. Zychowicz : L. Buzanska (*) Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego St., 02-106 Warsaw, Poland e-mail: buzanska@imdik.pan.pl L. Sirghi Department of Physics, Alexandru Ioan Cuza University, blvd. Carl I, 11, Iasi 700506, Romania Present Address: A. Ruiz Department of Applied Biotechnology and Translational Medicine, University of Milan, Via Vanvitelli 32, 20139 Milan, Italy Biomed Microdevices (2013) 15:495–507 DOI 10.1007/s10544-013-9749-9