Issue 6, 2010

The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening

Abstract

We present a rapid, reproducible and sensitive neurotoxicity testing platform that combines the benefits of neurite outgrowth analysis with cell patterning. This approach involves patterning neuronal cells within a hexagonal array to standardize the distance between neighbouring cellular nodes, and thereby standardize the length of the neurite interconnections. This feature coupled with defined assay coordinates provides a streamlined display for rapid and sensitive analysis. We have termed this the network formation assay (NFA). To demonstrate the assay we have used a novel cell patterning technique involving thin film poly(dimethylsiloxane) (PDMS) microcontact printing. Differentiated human SH-SY5Y neuroblastoma cells colonized the array with high efficiency, reliably producing pattern occupancies above 70%. The neuronal array surface supported neurite outgrowth, resulting in the formation of an interconnected neuronal network. Exposure to acrylamide, a neurotoxic reference compound, inhibited network formation. A dose–response curve from the NFA was used to determine a 20% network inhibition (NI20) value of 260 µM. This concentration was approximately 10-fold lower than the value produced by a routine cell viability assay, and demonstrates that the NFA can distinguish network formation inhibitory effects from gross cytotoxic effects. Inhibition of the mitogen-activated protein kinase (MAPK) ERK1/2 and phosphoinositide-3-kinase (PI-3K) signaling pathways also produced a dose-dependent reduction in network formation at non-cytotoxic concentrations. To further refine the assay a simulation was developed to manage the impact of pattern occupancy variations on network formation probability. Together these developments and demonstrations highlight the potential of the NFA to meet the demands of high-throughput applications in neurotoxicology and neurodevelopmental biology.

Graphical abstract: The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening

Supplementary files

Article information

Article type
Paper
Submitted
23 Oct 2009
Accepted
07 Dec 2009
First published
04 Jan 2010

Lab Chip, 2010,10, 701-709

The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening

J. Frimat, J. Sisnaiske, S. Subbiah, H. Menne, P. Godoy, P. Lampen, M. Leist, J. Franzke, J. G. Hengstler, C. van Thriel and J. West, Lab Chip, 2010, 10, 701 DOI: 10.1039/B922193J

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