Microfluidic construction of minimalistic neuronal co-cultures

Abstract

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems).The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency E patt was .75% during lengthy in chip culture, with y85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

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.…”

Preparation of Neuronal Co-cultures with Single Cell Precision

“…The use of lengthy treatments vaporizes the water mask, leading to excessive plasma disintegration of the polyamine coating. On occasion insufficient plasma treatment results, leaving intact polyamine coatings on the adjacent microchannel wall (see Figure 3D) that can support neuron adhesion 12 . Nevertheless, arrayed mouse cortical neurons had a patterning efficiency of 89.5% 12 following culture for 6 days.…”

“…As such it is recommend that microfabrication of the SU-8 masters used for PDMS replication is outsourced to commercial or institutional facilities. The two layer mask designs are freely available as supplementary information (ZIP) on the Royal Society of Chemistry website 12 . Importantly, the first SU-8 layer should be fabricated to a depth of 2.5-3.0 μm, and the second to a depth of 25-30 μm.…”

“…Microfluidic arraying in combination with an in situ biomaterial patterning method can be used for the registration of highly interconnected neuronal co-cultures using minimal cell numbers. Microfluidic arraying is based on a differential flow approach [12][13][14][15] , whereby microstructured traps are positioned within a fluidic circuit (illustrated along with SEM images in Figure 1). The path 0 → 1 has the lower fluidic resistance (R 2 > R 1 ) for transporting neurons to a linear array of microstructured apertures -the inlets to the neurite outgrowth channels.…”

Acrylamide alters neurotransmitter induced calcium responses in murine ESC-derived and primary neurons

“…Isolation of pCN from C57BL/6N mice at embryonic day 16 was conducted as described in Dinh et al (2013;see suppl. material).…”