A High-Throughput Assay of Cell-Surface Interactions using Topographical and Chemical Gradients By Jing Yang, Felicity R.A. J. Rose, Nikolaj Gadegaard, and Morgan R. Alexander* Biomaterials are a significant part of modern healthcare; with intraocular lenses, coronary stents, degradable sutures, catheters, vascular grafts and hip replacements all commonplace. The surface properties of man-made materials are known to dictate biological response in vitro, and it is thought that in many cases this has relevance to in vivo performance. [1–3] Current biomaterial applications and emerging areas such as tissue engineering, require a fuller understanding of cell-surface interactions. Measurements of cellular response to surfaces including adhesion, function, proli- feration, migration and differentiation have been shown to be modulated by the surface chemistry, [4–9] topography, [10–15] and elasticity [16,17] of materials. Conventional studies of cell-surface interactions are carried out by varying each individual surface pro- perty using separate samples whilst keeping other surface properties constant. The use of gradient format samples has been encouraged, nevertheless, by the highly illustrative work of Chaudhury and Whitesides in the early 90s, where self-assembled chemical gradients of sufficiently steep wettability changes were used to move sessile water droplets uphill. [18] Recently, single- gradient sample formats containing a range of roughnesses, arginine-glycine-aspartic acid (RGD)-peptide densities, and wettabi- lities have been used to investigate cell adhesion depending on each of these parameters on one sample. [19–21] Combinatorial sample formats, such as microarray polymer libraries [22–24] and orthogonal- gradient sample formats [25,26] have been reported for the investigation of cell adhesion and morphology. Microarray polymer libraries allow parallel assessment of hundreds of polymers of distinctly different types, while the orthogonal-gradient sample format lends itself to the investigation of a combination of two surface parameters in a continuously variable format, where modulation from two extremes occurs in both axes by very small steps. The orthogonal-gradient approach reduces the large number of samples and experiments that would be required to investigate all possible combinations of two surface properties. This opens the possibility of using high-throughput readouts of cell response. Previous studies using a poly (e-caprolactone)-poly(D,L-lactide) polymer blend have reported achieving a chemical gradient by varying the bulk composition in one direction whilst applying a gradient of temperature orthogonally, to form topographical features through phase separation. [25] Orthogonal gradients have also been achieved by varying the molecular weights of two polymers and the grafting density. [26] Both of these approaches are limited in that the microstructures formed are determined by the polymer behaviour. Here, we present an orthogonal-gradient system suitable for the production of a wide range of topographies achievable through embossing and the large selection of chemistry offered by plasma polymerization. To prove this concept we use conformal plasma polymer coatings on micropatterned polymer substrates, choosing a sur- face chemistry previously found to have application in tissue engineering [6,7] and groove topographies that have been shown to strongly order cells shape. [10] Plasma polymer deposition allows control of surface chemistry independent of the substrate. [27] High-resolution pattern design combined with hot embossing can be used to make substrates with topographical features defined by the experimentalist with great precision and flexibility. [28–30] The two gradients are oriented perpendicularly on a poly(methyl methacrylate) (PMMA) substrate, as shown in Figure 1A. Each gradient varies continuously over a distance of 10 mm. The large number of combinations of surface chemistry and topography that are present on the sample can be charac- terised using analysis techniques under automated acquisition, such as x-ray photoelectron spectroscopy (XPS) and water contact- angle measurements. [23,31] Cell response to the sample is captured using automated fluorescence microscopy. We study fibroblast adhesion and orientation to illustrate how this com- binatorial sample format can be coupled with high-throughput analysis techniques, to identify surface property–cellular response relationships. The topographical gradient consisted of parallel grooves with widths varying from 5 mm to 95 mm, separated by 5 mm wide ridges shown schematically in Figure 1B. Hot embossing of a thin (0.5 mm) PMMA sheet against a patterned silicon master was used to faithfully reproduce the topography of the master. In the direction perpendicular to the topographical gradient, a wett- COMMUNICATION www.advmat.de [*] Dr. M. R. Alexander, Dr. J. Yang Laboratory of Biophysics and Surface analysis, School of Pharmacy University of Nottingham University Park, Nottingham, NG7 2RD (UK) E-mail: morgan.alexander@nottingham.ac.uk Dr. F. R. A. J. Rose Division of Advanced Drug Delivery & Tissue Engineering, School of Pharmacy Centre for Biomolecular Sciences University of Nottingham University Park, Nottingham, NG7 2RD (UK) Dr. N. Gadegaard Centre for Cell Engineering Department of Electronics and Electrical Engineering Glasgow University, University Avenue, Glasgow, G12 8LT (UK) DOI: 10.1002/adma.200801942 300 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2009, 21, 300–304