The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening
The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening
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 mu M. 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.
acid-induced differentiation, human neuroblastoma-cells
neural stem-cells, in-vitro developmental neurotoxicity, surface-chemistry, neuronal networks, soft lithography, cultured neurons, patterned growth
701-709
Frimat, J.P.
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Sisnaiske, J.
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Subbiah, S.
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Menne, H.
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Godoy, P.
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Lampen, P.
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Leist, M.
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Franzke, J.
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Hengstler, J.G.
ae4a2b59-c041-4fca-9a66-d0d6a7053371
van Thriel, C.
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West, J.
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4 January 2010
Frimat, J.P.
e8bb0e6a-729b-48bf-8a3b-5c30f9878832
Sisnaiske, J.
153da33a-b334-450e-b7f0-2c6942999ac7
Subbiah, S.
6c4b68dc-f292-4b3d-8d96-c1e6a1893503
Menne, H.
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Godoy, P.
3b9d5541-7613-4dbb-99c6-b8ee1e68ef8f
Lampen, P.
4be26759-967c-4ca3-85d8-189fdd9ddba3
Leist, M.
99b41384-9c01-438f-b1c3-d67ed6a0e062
Franzke, J.
fd24d4c2-387e-4d2d-981c-dcf045aaf41e
Hengstler, J.G.
ae4a2b59-c041-4fca-9a66-d0d6a7053371
van Thriel, C.
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West, J.
f1c2e060-16c3-44c0-af70-242a1c58b968
Frimat, J.P., Sisnaiske, J., Subbiah, S., Menne, H., Godoy, P., Lampen, P., Leist, M., Franzke, J., Hengstler, J.G., van Thriel, C. and West, J.
(2010)
The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening.
Lab on a Chip, 10 (6), .
(doi:10.1039/B922193J).
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 mu M. 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.
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More information
Published date: 4 January 2010
Additional Information:
ISI Document Delivery No.: 567UB
Times Cited: 26
Cited Reference Count: 56
Frimat, Jean-Philippe Sisnaiske, Julia Subbiah, Subanatarajan Menne, Heike Godoy, Patricio Lampen, Peter Leist, Marcel Franzke, Joachim Hengstler, Jan G. van Thriel, Christoph West, Jonathan
European Community's [FP7/2007-2013, HEALTH-F5-2008-201619]; Deutsche Forschungsgemeinschaft [WE 3737/3-1]; Ministerium fur Innovation, Wissenschaft, Forschung und Technologie des Landes Nordrhein-Westfalen; Bundesministerium fur Bildung und Forschung
The authors are grateful to Uli Marggraf for SU-8 master fabrication, Norman Ahlman for white light interferometry, Maria Becker for SEM analysis, and Sasidhar Maddula and Cristina Cadenas for providing cell types. Financial support from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. HEALTH-F5-2008-201619 (ESNATS), the Deutsche Forschungsgemeinschaft (WE 3737/3-1), the Ministerium fur Innovation, Wissenschaft, Forschung und Technologie des Landes Nordrhein-Westfalen and from the Bundesministerium fur Bildung und Forschung is also gratefully acknowledged.
Royal soc chemistry
Cambridge
Keywords:
acid-induced differentiation, human neuroblastoma-cells
neural stem-cells, in-vitro developmental neurotoxicity, surface-chemistry, neuronal networks, soft lithography, cultured neurons, patterned growth
Organisations:
Cancer Sciences
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Local EPrints ID: 346443
URI: http://eprints.soton.ac.uk/id/eprint/346443
ISSN: 1473-0197
PURE UUID: a916e0b3-4e87-4f0d-8877-011fa3e09d36
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Date deposited: 28 Jan 2013 11:26
Last modified: 15 Mar 2024 03:43
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Author:
J.P. Frimat
Author:
J. Sisnaiske
Author:
S. Subbiah
Author:
H. Menne
Author:
P. Godoy
Author:
P. Lampen
Author:
M. Leist
Author:
J. Franzke
Author:
J.G. Hengstler
Author:
C. van Thriel
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