The authors are grateful to Ulrich Marggraf (ISAS) for SU-8 fabrication and Maria Becker (ISAS) for SEM imaging. The research was financially supported by the Deutsche Forschungsgemeinschaft (DFG WE3737/3-1), a Bundesministerium f&#252;r Bildung und Forschung grant (BMBF 0101-31P6541) and by the Ministerium f&#252;r Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen. Heike Hardelauf thanks the International Leibniz Graduate School "Systems Biology Lab-on-a-Chip" for financial support.

The protocols are intended for neuroscientists, and are summarized in Figure 2. 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 website12. Importantly, the first SU-8 layer should be fabricated to a depth of 2.5-3.0 &#956;m, and the second to a depth of 25-30 &#956;m. These are k...

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The microfluidic arraying technique is the first of its kind that enables precision single neuron handling for establishing minimalistic cultures. Coupled with the in situ biomaterial patterning method, a powerful approach to align cell patterns with microfluidic structures, these minimalistic cultures have high intercompartment connectivity levels with reduced local entanglement. These features can be used for the efficient study of inter-compartment transmission, and with simple design modifications have the p...

Neuronal tissue is highly complex; a heterogeneous cellular mixture that is spatially ordered within defined layers and compartments and with plastic connectivity via cell contacts and especially via axon and dendrite outgrowths. New techniques are required to confer greater experimental freedom to gain deeper insights and unravel mechanisms central to disease, development, and healthy function. The Campenot chamber1,2 and more recently microfabricated embodiments3,4 can be used for the ex vivo preparation of networked neuronal co-cultures with the ability to selectively perturb the different somatic populations and also their neurite outgrowths. These microfluidic devices have for example been used to study axon degeneration and regeneration following chemical5,6 or laser axotomy6-8, tauopathy9, viral dissemination10,11, and mRNA localization in axons4.

To extend the reach of the neurobiologists, technological developments are required to prepare minimalistic neuronal co-cultures. This enables disentanglement of the neuronal network for the investigation of the system with single cell and sub-cellular precision. The requirement for minimal cell numbers opens the possibility to analyze rare cell types, including dopaminergic substantia nigra cells relevant to Parkinson&#39;s disease, spiral ganglia from the ear, peripheral neurons, and stem cells. Beyond this, cellular economy is relevant to the 3Rs initiative. Using these microfluidic platforms, large-scale toxicity screens or other high throughput, data rich experimental series requiring animal neurons can now be considered.

In this paper protocols for the fabrication and use of a microfluidic device are described. 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 approach12&#8211;15, whereby microstructured traps are positioned within a fluidic circuit (illustrated along with SEM images in Figure 1). The path 0 &#8594; 1 has the lower fluidic resistance (R2 &#62; R1) for transporting neurons to a linear array of microstructured apertures &#8211; the inlets to the neurite outgrowth channels. Occupancy of the trap by a single cell locally impedes the flow to divert the streamlines for trapping subsequent cells in neighboring traps. Complete occupancy of the traps in the array switches the fluidic ratio (R1 &#62; R2) to divert the streamlines into the serpentine path (0 &#8594; 2) to produce a bypass mode of operation for the removal of excess neurons.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51389/51389fig1highres.jpg" width="500" /> 
Figure 1. Microfluidic Circuit. A) The differential resistance fluidic circuit for single neuron arraying, with flanking culture chambers interconnected by neurite outgrowth channels. B,C) SEM images of the bilayer compartmentalized neuron co-culture array with meniscus pinning micropillars. With this design, trident-shaped neuron trapping structures were used to promote fasciculation of the neurite outgrowths. Figure and legend reproduced with permission of the Royal Society of Chemistry (RSC)12. <a href="https://www.jove.com/files/ftp_upload/51389/51389fig1highres.jpg" target="_blank">Click here to view larger image.</a>
The preparation of micropatterned neuronal networks on planar substrates can readily be achieved (for examples from our group, see Frimat et al.16 and Heike et al.17). However, encapsulating bioactive material patterns within PDMS devices and with the requirement of micrometer-scale alignment of these to the microfluidic channels poses a major technical challenge. In section 3.1 a protocol for the in-chip, or in situ, preparation of biomaterial patterns is presented. These patterns enable neuron registration during lengthy culture timescales and promote outgrowths between compartments. Meniscus-pinning microstructures are used to align a so-called water mask with the neuron arraying sites and neurite outgrowth channels. The water mask protects adhesion molecule coatings during plasma treatment, whereas exposed surfaces are disintegrated to define the biomaterial pattern. In addition, protocols are provided for cell culture and for fluidic isolation necessary for the selective treatment of the different co-culture compartments.
The protocols are designed to leverage the user-friendly principles of soft lithography for the replication of poly(dimethylsiloxane) (PDMS) microfluidic devices18. Similarly, in situ biomaterial patterning is straightforward, exploiting evaporation and surface tension phenomena, and only requiring an inexpensive hand-held plasma source. The microfluidic circuit effectively programs cell loading and compartment specific treatments making these operations simply a matter of dispensing materials into the correct bottom port and aspirating from above. In this manner, it is intended to give the neurobiologists the freedom to prepare and use microfluidic devices in their own laboratories.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PDMS Sylgard 184</td>
			<td style="width:163px;">Dow Corning</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDMS Elastosil RT 601</td>
			<td style="width:163px;">Wacker</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Coverslips</td>
			<td style="width:163px;">VWR</td>
			<td>630-1590</td>
			<td>130-160 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">3 mm Biopsy Punches</td>
			<td style="width:163px;">Kai Medical</td>
			<td style="width:143px;"></td>
			<td>Handle with care - extremely sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon Tubing</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:143px;">S-50-HL</td>
			<td>1.65 mm ID; 3.35 mm OD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Syringe (Inkjekt and Omnifix)</td>
			<td style="width:163px;">Braun</td>
			<td style="width:143px;">6064204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">4-Way Tubing Connector</td>
			<td style="width:163px;">VWR or Fisher Scientific</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Flow Regulator</td>
			<td style="width:163px;">Harvard Apparatus</td>
			<td style="width:143px;">722645</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">0.5 mm Pins</td>
			<td style="width:163px;">Dressmaking Departments</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">PLL-g-PEG</td>
			<td style="width:163px;">SuSoS</td>
			<td style="width:143px;">PLL(20)-g[3.5]-PEG(5)</td>
			<td>Stability in storage can be an issue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-D-lysine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-lysine-FITC</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P3069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-ornithine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">fibronectin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">laminin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PBS</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Inverted Fluorescent Microscope</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Example Aspiration Pump</td>
			<td style="width:163px;">KNF Neuberger, Laboport</td>
			<td style="width:143px;">N811KVP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Hand Held Corona Discharge Device</td>
			<td style="width:163px;">Leybold-Heraeus, USA</td>
			<td style="width:143px;">VP23</td>
			<td>May not comply with your country&#39;s safety standards</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Femto Plasma Oven</td>
			<td style="width:163px;">Diener Electronic</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Vacuum Dessicator or Centrifuge</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of neuronal co-cultures with single cell precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Water masking exploits surface tension. The pressure tolerance at the air-liquid interface scales inversely with the radius of curvature [<img fo:content-width="1in" src="/files/ftp_upload/51389/51389eq1.jpg" width="113" />, producing highly stable interfaces at microfluidic dimensions. The micropillars effectively anchor the water mask in place. Water mask formation is driven by evaporation from the microfluidic ports, with the rate increased by heating. Using the heat from low magnification (4X - 10X) microscopy illumi...

Watch this Scientific Journal Video about Preparation of Neuronal Co-cultures with Single Cell Precision at JoVE.com

Preparation of Neuronal Co-cultures with Single Cell Precision

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

preparation-of-neuronal-co-cultures-with-single-cell-precision

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