Modulating patterned adhesion and repulsion of HEK 293 cells on microengineered parylene-C/SiO 2 substrates
Centre for Integrative Physiology, School of Biomedical Sciences, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, United Kingdom. . Journal of Biomedical Materials Research Part A
(Impact Factor: 3.37).
02/2013; 101A(2). DOI: 10.1002/jbm.a.34329
This article describes high resolution patterning of HEK 293 cells on a construct of micropatterned parylene-C and silicon dioxide. Photolithographic patterning of parylene-C on silicon dioxide is an established and consistent process. Activation of patterns by immersion in serum has previously enabled patterning of murine hippocampal neurons and glia, as well as the human hNT cell line. Adapting this protocol we now illustrate high resolution patterning of the HEK 293 cell line. We explore hypotheses that patterning is mediated by transmembrane integrin interactions with differentially absorbed serum proteins, and also by etching the surface substrate with piranha solution. Using rationalized protein activation solutions in place of serum, we show that cell patterning can be modulated or even inverted. These cell-patterning findings assist our wider goal of engineering and interfacing functional neuronal networks via a silicon semiconductor platform. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.
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Available from: Kevin M Shakesheff
- "Previous work has shown that parylene-C can facilitate protein adsorption and guide neuronal growth and neuronal network formation on parylene-C stripes deposited on a silicon oxide substrate . Cell types that have been patterned with parylene-C include murine hippocampal neurons and glia  , human teratocarcinoma (hNT) cell line derived neurons  and astrocytes , and HEK 293 cells . Cell patterning techniques have considerable impact in tissue engineering, as they enable detailed studies of the extracellular microenvironment and may lead to directed stem cell differentiation . "
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ABSTRACT: Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning.
Available from: Sylvain Vedraine
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ABSTRACT: Surface modification technology has made significant advances in recent years towards the miniaturization and organization of traditional cell culture systems. However, the capability of directing transfected cells and neuronal connections to probe small structures such as spines is still under development. In the current work, interactions of different micropatterned substrates with HEK 293, CF10 cell lines, and primary neuronal cultures are evaluated. Using conventional and confocal fluorescence microscopies, several morphological and behavioral aspects of all three cell types were investigated. The immortalized cell lines were able to attach to the substrate and interact with neighboring cells. Similarly, cortical neurons formed connections guided by the micropatterns. Transfection of HEK 293 or CF10 cell lines with specific members of the G protein-coupled receptor family did not alter the behavior of these cells in the micropatterns. On the other hand, neuronal projections were efficiently isolated by the patterns, simplifying the localization of spines with nano-scale resolution probed by atomic force microscopy. This work presents a valuable approach to isolate cells or to constrain important cell structures to grow along a desired pattern, thus facilitating advanced biological studies.
Available from: Mark A Hughes
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ABSTRACT: Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based 'lab on a chip' technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2 wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.
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