High fidelity neuronal networks formed by plasma masking with a bilayer membrane: analysis of neurodegenerative and neuroprotective processes

Abstract

Spatially defined neuronal networks have great potential to be used in a wide spectrum of neurobiology assays. We present an original technique for the precise and reproducible formation of neuronal networks. A PDMS membrane comprising through-holes aligned with interconnecting microchannels was used during oxygen plasma etching to dry mask a protein rejecting poly(ethylene glycol) (PEG) adlayer. Patterns were faithfully replicated to produce an oxidized interconnected array pattern which supported protein adsorption. Differentiated human SH-SY5Y neuron-like cells adhered to the array nodes with the micron-scale interconnecting tracks guiding neurite outgrowth to produce neuronal connections and establish a network. A 2.0 μm track width was optimal for high-level network formation and node compliance. These spatially standardized neuronal networks were used to analyse the dynamics of acrylamide-induced neurite degeneration and the protective effects of co-treatment with calpeptin or brain derived neurotrophic factor (BDNF).

Micropatterning neuronal networks

“…The SU-8 master fabrication and PDMS replication methods have previously been described. 10 Briey, bilayer plasma stenciling involves conformally contacting the stencil with the substrate coated with the cytophobic material and plasma etching (70 W, 40 kHz (Femto, Diener Electronic)) in a 0.2 mbar oxygen atmosphere for 60 s. The exposed substrate regions were backlled with polylysine (100 mg mL À1 in 1Â PBS) for 15 minutes, followed by washing with 1Â PBS or culture medium for 1 hour. Alternatively, backlling was investigated using aminosilanes purchased from ABCR, Germany; 3-aminopropyl triethoxysilane (APTES), diethylenetriaminosilane (DETA), bis(trimethoxy silylpropyl)amine (BTMSPA) and 3-aminopropyldiisopropylethoxysilane (APDIPES).…”

“…[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

“…PEGylated surfaces resist SH-SY5Y cell adhesion. 8 In a preliminary experiment using PL/PLL-g-PEG patterns on planar glass substrates differentiated SH-SY5Y cells had a patterning efficiency of 87% after 5 DIV culture (ESI3, Fig. 3).…”

“…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.…”

Developmental neurotoxicity: the case of perfluoroalkylated compounds

“…In this context, it seems to be relevant that perfluoroalkylated compounds affect protein kinase C signalling that is known as a key factor of cell migration (Stewart et al 2012), a process of particular relevance during the development of the nervous system. Neurotoxicity (Takahashi et al 2011;Nakamura et al 2011;Li et al 2011;Sriram et al 2010;Liu et al 2010) as well as developmental neurotoxicity (Pamies et al 2010;Frimat et al 2010;Hardelauf et al 2011;Kadereit et al 2012;Kuegler et al 2010;Hartung et al 2011) represent cutting-edge topics in toxicology, reflected by the particularly high number of articles in this field. However, little has been published discussing the neurotoxicity of perfluoroalkylated compounds, although they are still widely used in pesticides, paints, clothes treatment, fire-fighting foams, carpets and leather products.…”