Engineering neuronal networks on photolithographically defined, biologically activated silicon substrates

Abstract Background To interface neurons with silicon semiconductors is a bioengineering challenge. Such constructs provide a platform for understanding neuronal coding and learning, an arena for modelling disease, and ultimately a step towards creating intelligent neuroprostheses. A prerequisite is the ability to dictate the spatial organisation of neuronal cells, in a context in which electrical and synaptic behaviour can be assessed. Our aim was to pattern cells on silicon, leading the way to the eventual incorporation of a wide range of biomicroelectromechanical systems for cellular interrogation. Methods A predetermined design of the chlorine-containing polmer parylene-C was photolithographically printed onto oxidised silicon wafers (in a microelectronics cleanroom facility). Parylene-patterned chips were activated by acid etching and then incubated in fetal calf serum. The resulting substrate became differentially cell-adhesive (parylene) or repulsive (SiO2) for certain cell types. A human glioma-derived stem-like cell line that had been isolated during debulking surgery was cultured on-chip, growing selectively on parylene. Neurons (Lund Human Mesencephalic [LUHMES] cell line) were then plated, adhering to the glial template and extending neurites to connect with adjacent cells to form a network. The effect of parylene design on neuronal network configuration was assessed by use of a pattern of circular nodes with four orthogonal spokes printed in arrays of three different configurations (node diameter 50 μm, spoke length 125 μm; node 100 μm, spoke 100 μm; node 250 μm, spoke 100 μm). Findings Ten different cell lines were assessed on the parylene–silicon cell-patterning platform. Cell adhesion behaviour was heterogeneous, with only select cell lines patterning at high resolution. LUHMES neurons in isolation failed to pattern. However, in co-culture with glioma-derived stem-like cells (which pattern accurately), the neurons adhered and differentiated to form networks. Neurite directionality differed significantly according to parylene design (Kolmogorov-Smirnov 250 μm vs 100 μm nodes D=0·204, p=0·001; 100 μm vs 50 μm nodes D=0·147, p=0·028), allowing creation of reticular neuronal networks. Interpretation The cell adhesion molecule profile of different cell lines affects their behaviour on the parylene–silicon platform. By consolidating DNA microarray data from cell types with contrasting patterning behaviour, we hope to correlate gene expression profiles with a cell's tendency to adhere to, or be repulsed by, the two contrasting substrates. The co-culture approach we describe has allowed creation of defined neuronal networks on silicon. The next steps include assessing long-term viability and functionality of patterned networks, while also improving control over their spatial organisation. Funding Wellcome Trust.