title: 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.
authors.affiliation: ISAS-Institute for Analytical Sciences, Otto-Hahn-Str. 6b, D-44227, Dortmund, Germany.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Frimat
authors.forename: Julia
authors.initials: J
authors.lastname: Sisnaiske
authors.forename: Subanatarajan
authors.initials: S
authors.lastname: Subbiah
authors.forename: Heike
authors.initials: H
authors.lastname: Menne
authors.forename: Patricio
authors.initials: P
authors.lastname: Godoy
authors.forename: Peter
authors.initials: P
authors.lastname: Lampen
authors.forename: Marcel
authors.initials: M
authors.lastname: Leist
authors.forename: Joachim
authors.initials: J
authors.lastname: Franzke
authors.forename: Jan G
authors.initials: JG
authors.lastname: Hengstler
authors.forename: Christoph
authors.initials: C
authors.lastname: van Thriel
authors.forename: Jonathan
authors.initials: J
authors.lastname: West
mesh_terms: D001681:Biological Assay / Q000295:instrumentation*; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002469:Cell Separation / Q000295:instrumentation*; D004305:Dose-Response Relationship, Drug; D004867:Equipment Design; D019544:Equipment Failure Analysis; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D009415:Nerve Net / Q000187:drug effects; D016501:Neurites / Q000187:drug effects* / Q000502:physiology; D009498:Neurotoxins / Q000633:toxicity*; D018675:Toxicity Tests / Q000295:instrumentation*
chemical_list: D009498:Neurotoxins