Modulating patterned adhesion and repulsion of HEK 293 cells on microengineered parylene-C/SiO(2) substrates.
ABSTRACT 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.
- SourceAvailable from: Kevin M Shakesheff[Show abstract] [Hide abstract]
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.Colloids and surfaces B: Biointerfaces 12/2014; 126. DOI:10.1016/j.colsurfb.2014.12.020 · 4.28 Impact Factor
- [Show abstract] [Hide abstract]
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.Journal of Visualized Experiments 03/2014; DOI:10.3791/50929
- [Show abstract] [Hide abstract]
ABSTRACT: Interfacing neurons with silicon semiconductors is a challenge being tackled through various bioengineering approaches. Such constructs inform our understanding of neuronal coding and learning and ultimately guide us towards creating intelligent neuroprostheses. A fundamental pre-requisite is to dictate the spatial organization of neuronal cells. We sought to pattern neurons using photolithographically defined arrays of polymer parylene-C, activated with fetal calf serum. We used a purified human neuronal cell line (Lund Human Mesencaphalic, LUHMES) to establish whether neurons remain viable when isolated on-chip or whether they require a supporting cell substrate. When cultured in isolation, LUHMES neurons failed to pattern and did not show any morphological signs of differentiation. We therefore sought a cell type with which to pre-pattern parylene regions, hypothesizing that this cellular template would enable secondary neuronal adhesion and network formation. From a range of cell lines tested, HEK 293 cells patterned with highest accuracy. LUHMES neurons adhered to pre-established HEK 293 cell clusters and this co-culture environment promoted morphological differentiation of neurons. Neurites extended between islands of adherent cell somata, creating an orthogonally arranged neuronal network. HEK 293 cells appear to fulfill a role analogous to glia, dictating cell adhesion and generating an environment conducive to neuronal survival. We next replaced HEK 293 cells with slower growing glioma-derived precursors. These primary human cells patterned accurately on parylene and provided a similarly effective scaffold for neuronal adhesion. These findings advance the use of this microfabrication-compatible platform for neuronal patterning.Journal of Biomedical Materials Research Part A 06/2013; 102(5). DOI:10.1002/jbm.a.34813 · 2.83 Impact Factor