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 (NI(20)) value of 260 microM. 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.

Micropatterning neuronal networks

“…[9][10][11][12] Ex vivo neuronal circuits can be constructed by restricting neurons within microchannel architectures [13][14][15] or, more commonly, by micropatterning an adhesive environment against a so-called cytophobic background that resists cell adhesion. The latter approach does not physically restrict neuron development, but instead provides spatially-dened biochemical guidance cues for the directed organisation of the neuronal circuit.…”

Microfluidic construction of minimalistic neuronal co-cultures

“…Great progress has been made with biomaterial patterning on planar substrates to spatially define cell cultures. [6][7][8][31][32][33][34][35] However, methods for aligned biomaterial patterning within microfluidic environments are poorly developed. The described CNA devices require biomaterial coatings on the neurons trapping sites to register the neurons.…”

“…For example, isolated single SH-SY5Y neuroblastoma cells do not ordinarily produce outgrowths. 6,8 This highlights a general lesson in that we often need to refine our engineering perspectives to best mimic neuronal tissues in vitro. To balance these interests an array system comprising isolated multi-neuron adhesion nodes is recommended.…”

Highlight report: acetaminophen hepatotoxicity

“…Currently, intensive research activities are ongoing to establish in vitro systems particularly for hepatotoxicity (Grinberg et al 2014;Ramaiahgari et al 2014;Rodrigues et al 2013;Schug et al 2013), nephrotoxicity (Sanchez-Niño et al 2014;Fujiki et al 2013;, neurotoxicity (Barbosa et al 2014;Sisnaiske et al 2014;Frimat et al 2010) and developmental toxicity (Krug et al 2013;Rempel et al 2015;Balmer et al 2014;Zimmer et al 2014). For validation of in vitro systems with hepatocytes APAP represents a popular reference compound (Jennings et al 2014;Hengstler et al 2014).…”