Cell patterning has emerged as an elegant tool in developing cellular arrays, bioreactors, biosensors, and lab-on-chip devices and for use in engineering neotissue for repair or regeneration. In this study, micropatterned surfaces were created using the layer-by-layer lift-off (LbL-LO) method for analyzing canine chondrocytes response to patterned substrates. Five materials were chosen based on our previous studies. These included: poly(dimethyldiallylammonium chloride) (PDDA), poly(ethyleneimine) (PEI), poly(styrene sulfonate) (PSS), collagen, and chondroitin sulfate (CS). The substrates were patterned with these five different materials, in five and ten bilayers, resulting in the following multilayer nanofilm architectures: (PSS/PDDA)5, (PSS/PDDA)10; (CS/PEI)4/CS, (CS/PEI)9/CS; (PSS/PEI)5, (PSS/PEI)10; (PSS/Collagen)5, (PSS/Collagen)10; (PSS/PEI)4/PSS, (PSS/PEI)9/PSS. Cell characterization studies were used to assess the viability, longevity, and cellular response to the configured patterned multilayer architectures. The cumulative cell characterization data suggests that cell viability, longevity, and functionality were enhanced on micropatterned PEI, PSS, collagen, and CS multilayer nanofilms suggesting their possible use in biomedical applications. 1. Introduction Replicating the highly structured in vivo microenvironment is crucial in understanding cellular behavior [1]. Traditional cell culture surfaces cannot provide sufficient control over the cellular microenvironment [2] for use in studying many anchorage-dependent cellular processes such as cellular differentiation, proliferation, and phenotypic expression. Cell supportive substrates, with the requisite spatiotemporal surface properties, are also a critical feature in designing appropriate biomaterial surfaces for use in cell arrays, bioreactors, biosensors [3], and cocultures [4–6] and for use in engineering new tissues for repair or replacement. Micropatterned surfaces have been explored as a means not only to answer fundamental questions in cell biology but also to develop cell culture substrates with surface features tailored for specific bio- and tissue engineering applications [2, 3, 7]. This was demonstrated by the growth of hepatocytes on micropatterned surfaces [4, 5]. The authors observed decreased DNA production and increased cellular apoptosis associated with a decrease in the adhesiveness of the surfaces [7]. Cell shape was also found to be the regulatory factor in both cell apoptosis and growth [7, 8]. This was achieved by an increasing restriction of the size of micropatterned
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