Recent development in lithography and surface treatment techniques have led to many advantages in the understanding of the mechanisms behind the interaction between cells and substrates due to the possibility to engineer chemical and topographical surface features up to the nanometer scale. Micropatterning use these technologies with the scope of encouraging cells to assume a specific and pre-determined shape, in order to study how this might influence cytoskeletal composition and features, along with cells apoptosis, migration and phenotype changes [5]. While soft lithography related techniques are among the most commonly used in research applications[6], other techniques are also being employed, such as inkjet printing [1], photochemistry [2] and laser-bioprinting [3]. While all of these methods are proven to be effective, they maintain an approximate resolution above the micrometer scale [4]. From previous studies it is known that human endothelial cells constrained on a micropattern can reach each other up to 20 μm distance trough a hydrophobic surface [4], but no further information could be found regarding these links, their nature and how they change in quality and quantity by variating the distance between cellularized patterns. This study aims to fill the gap by coupling high resolution patterning techniques and immunostaining in order to better understand the dynamic of endothelial cell-to cell interaction with a special focus on how the horizontal distances affect this mechanism. The method we propose combine electron beam lithography technique and binary gas-phase silanization on glass featuring engineered nano-scale characteristics. The final results will see a pattern made of a silane self-assembled-monolayers (SAM) having cell-adhesive characteristics on a cell-repulsive SAM background. In order to validate the protocol’s efficiency for the creation of micropatterns, Human Umbilical Vein Endothelial Cells (HUVECs) are cultured on the binary substrates and observation on their cytoskeleton and phenotype are been performed. References: [1] Gauvreau V, Laroche G. Micropattern printing of adhesion, spreading, and migration peptides on poly(tetrafluoroethylene) films to promote endothelialization. Bioconjug Chem. 2005; 16:108897. [PubMed:16173784] [2] Barbucci R, Lamponi S, Magnani A, Pasqui D. Micropatterned surfaces for the control of endothelial cell behaviour. Biomol Eng. 2002; 19:161–70. [PubMed: 12202177] [3] Guillemot F, Souquet A, Catros S, Guillotin B, Lopez J, Faucon M, Pippenger B, Bareille R, Remy M, Bellance S, Chabassier P, Fricain JC, Amedee J. High-throughput laser printing of cells and biomaterials for tissue engineering. Acta Biomater. 2010; 6:2494–500. [PubMed: 19819356] [4] Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Micropatterned surfaces for control of cell shape, position, and function. Biotechnol Prog. 1998; 14:356–63. [PubMed: 9622515] [5] Anderson, D.E.J., Hinds, M.T. Endothelial Cell Micropatterning: Methods, Effects, and Applications. Ann Biomed Eng 39, 2329–2345 (2011). https://doi.org/10.1007/s10439-011-0352-z [6] Fink J, Théry M, Azioune A, et al. Comparative study and improvement of current cell micro-patterning techniques. Lab Chip. 2007;7(6):672-680. doi:10.1039/b618545b]