Abstract
Controlling the spatial distribution of glia and neurons in in vitro culture offers the opportunity to study how cellular interactions contribute to large scale network behaviour. A recently developed approach to cell-patterning uses differential adsorption of animal-serum protein on parylene-C and SiO2 surfaces to enable patterning of neurons and glia. Serum, however, is typically poorly defined and generates reproducibility challenges. Alternative activation methods are highly desirable to enable patterning without relying on animal serum. We take advantage of the innate contrasting surface chemistries of parylene-C and SiO2 to enable selective bonding of polyethylene glycol SiO2 surfaces, i.e. PEGylation, rendering them almost completely repulsive to cell adhesion. As the reagents used in the PEGylation protocol are chemically defined, the reproducibility and batch-to-batch variability complications associated with the used of animal serum are avoided. We report that PEGylated parylene-C/SiO2 substrates achieve a contrast in astrocyte density of 65:1 whereas the standard serum-immersion protocol results in a contrast of 5.6:1. Furthermore, single-cell isolation was significantly improved on PEGylated substrates when astrocytes were grown on close-proximity parylene-C nodes, whereas isolation was limited on serum-activated substrates due tolerance for cell adhesion on serum-adsorbed SiO2 surfaces.
Highlights
Patterned cultures offer the opportunity to study cell communication at the cellular and network level
While contrast in cell adhesiveness was observed for the two PEGylation control substrates there was a significant amount of cell adhesion to the SiO2 regions
We report that the surface of our parylene-C substrates contained approximately 2% oxygen after piranha acid treatment, determined by x-ray photoelectron spectroscopy, Fig. 4a demonstrates this does not appear to result in significant PEGylation of the parylene-C surface and did not affect astrocyte adhesion to parylene-C
Summary
Patterned cultures offer the opportunity to study cell communication at the cellular and network level. Of particular interest to the neuroscience community, the precise placement of both neurons and glia enables study of how the behaviour of single cells contribute larger scale network behaviour. One cell-patterning technique that holds promise is the parylene-C/SiO2 platform developed by Delivopoulos et al.[1]. The platform utilises the biocompatible polymer parylene-C deposited onto a SiO2 background to create surfaces that are attractive and repulsive to cell adhesion, respectively. While a multitude of cell-patterning techniques are available[2], the parylene-C/SiO2 platform is attractive for neuroscience research as it is integrated into multi-electrode arrays. Unsworth et al subsequently used the platform to isolate single astrocytes on nodes of parylene[4] In both these works, the glia displayed a limited tendency to grow into normally cell-repulsive SiO2 regions. Delivopoulos et al and Hughes et al hypothesize that cell patterning is the result of the combinatorial effects resulting from the adsorption of specific serum proteins and the proteins conformation once adsorbed[1,9,10]
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