Name: Preparation of Neuronal Co-cultures with Single Cell Precision (Video) | JoVE Journal
Uploaded: 2014-05-20T13:00:00.000+00:00
Duration: 9 min 6 s
Description: Learn to prepare interconnected neuronal co-cultures with single cell precision using microfluidic arraying and plasma patterning techniques. This method enables efficient neuroscience research with minimal cell inputs.

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5 minutes

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 μm, and the second to a depth of 25-30 μm. These are key dimensions necessary for effective neuron arraying. The 3 micron height limit is also necessary to prevent neurons being transported or migrating between the two compartments12.
1. Microfluidic Device Replication in PDMS
<ol>
	<li>Mix the PDMS prepolymer thoroughly with the curing agent (184) at a ratio of 10:1 and degas the mixture in a vacuum desiccator for typically 20 min. Alternatively, put the mixture in a 50 ml Falcon tube and use low g centrifugation for degassing.</li>
	<li>Place the 2-layer microstructured SU-8 wafer on an 80 °C hotplate and align polymer frames to each device on the wafer. Pour PDMS into the frames to a depth of ≥5 mm and allow &gt;1 hr to ensure complete thermal curing.</li>
	<li>Once cured, remove the wafer from the hotplate and allow to cool on a flat surface.</li>
	<li>Wear protective eyewear and work from one corner (away from the SU-8 microstructures) with a rigid scalpel to gently prize the frame and PDMS from the wafer.</li>
	<li>Remove the PDMS mold from the frame and use a pair of scissors to trim the device of excess PDMS. This so-called flashing is often produced when a thin film of PDMS spreads between the polymer frame and the wafer surface.</li>
	<li>Remaining PDMS can be applied to the entire wafer and cured to protect the wafer during storage. Remove this prior to subsequent device molding, and in doing so remove any particulate contaminants from the surface.</li>
</ol>
2. Device Assembly and Interconnection
<ol>
	<li>Prepare the 6 interface ports using a 3 mm diameter biopsy punch. Work on a dark surface, in good light conditions and with the microstructured features facing upwards to aid alignment of the ports with the microfluidic channels.</li>
	<li>Inspect the device for particulates. Remove these using reversible adhesive tape.</li>
	<li>The microfluidic circuit is encapsulated using a thin PDMS layer mounted on a glass coverslip: These are prepared by pouring a small volume (~0.5 ml) of the PDMS-curing agent mixture (10:1, 601) onto the base of a flexible, bacteriological grade Petri dish. Gently press a coverslip onto the PDMS layer, place on the hotplate and thermally cure for a few minutes. Quickly after cooling, use a scalpel to cut out and prize the coverslip-PDMS bilayer from the Petri dish. Trim with scissors if necessary.</li>
	<li>Plasma bond the PDMS device and support layer to produce a good seal: Optimum conditions are instrument specific. For example, a plasma oven operating at 70 W and 40 kHz in a 0.2 mbar oxygen atmosphere for 40 sec produces an excellent PDMS–PDMS bond. Once plasma treated, press both parts together and leave for a minute to fully bond.</li>
	<li>Use silicone tubing (1.6 mm ID; 3.2 mm OD) to interface with pumps and syringes.</li>
</ol>
3. Biomaterial Patterning and Microfluidic Operations
The protocols for in situ biomaterial patterning, neuron culture and fluidic treatments are illustrated in Figure 2.
<img alt="Neuron array process diagram; bioadhesive coating, plasma patterning, PBS wash, PLL-g-PEG." fo:content-width="5in" src="/files/ftp_upload/51389/51389fig2highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Illustrated microfluidic protocols. A) PL and PLL-g-PEG patterning; addition of the cell adhesion layer (e.g., PL, green), with evaporation producing the aligned water mask. B) Atmospheric air plasma treatment of the exposed PL; two antenna pins are used, with the plasma introduction antenna illustrated with a pink star. C) PBS (blue) washing by aspiration is used to avoid contaminating the plasma-treated region with PL. D) Delivery of PLL-g-PEG (red) by aspiration to coat the plasma-treated regions. E) Cell loading; simultaneous microfluidic arraying of neurons to both flanking compartments by aspiration, F) media (pink) perfusion using a hydrostatic-driven flow. G) Isolated fluidic treatments; peripheral treatment of a test agent (black) delivered from the bottom flanking inlet by aspiration and H) central treatment delivered from the bottom central inlet by aspiration. Figure and legend reproduced with permission of the Royal Society of Chemistry (RSC)12. <a href="https://www.jove.com/files/ftp_upload/51389/51389fig2highres.jpg" target="_blank">Click here to view larger image.</a>
Immediately following plasma bonding the PDMS microfluidic channels are highly hydrophilic (contact angle ≤5°). Such surfaces are suitable for the adhesion and culture of cell lines such as differentiated human SH-SY5Y neuron-like cells. For the ex vivo culture of primary neurons and the culture of neuronal precursor cell lines such as the Lund human mesencephalic cell line (LUHMES) a polyamine coating, either polylysine (PL) or polyornithine (PO), is required as an electrostatic anchor. In addition adhesion proteins such as laminin or fibronectin are often required as the integrin-interfacing coating. However, the PL and PO coatings readily bind neurons, hindering the microfluidic delivery to the array sites and also imposing unwanted shear stresses. To solve this problem in-chip biomaterial patterning is required to localize adhesion materials at the trap sites and the outgrowth microchannels. This also promotes the development of inter-compartment connections and reduces local entanglement. The protocol also involves the addition of PEG materials to neighboring areas to ensure proteins and neurons are restricted to the desired locations during lengthy culture. The following protocol is documented in Figure 3:
<img alt="Microfluidic chip design and fluorescence microscopy analysis; diagrams and images in research study." fo:content-width="5in" src="/files/ftp_upload/51389/51389fig3highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Water masking for biomaterial patterning by plasma stenciling. A) Illustration of the water masking process with the PDMS micropillars pinning the water meniscus in place for plasma stenciling. Curvature of the meniscus in the vertical plane has been neglected. B) Water mask visualized with a red dye and positioned by meniscus pinning. C) The patterned PL-FITC coating imaged after plasma stenciling. D) Addition of protein rejecting and cell repellent PLL-g-PEG–TRITC to a plasma-patterned PL-FITC coating. This was used for the registration of SH-SY5Y cells and also for patterning an additional fibronectin layer for LUHMES cells. Figure and legend reproduced with permission of the Royal Society of Chemistry (RSC)12. <a href="https://www.jove.com/files/ftp_upload/51389/51389fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<ol>
	<li>Water Masking for In situ Biomaterial Patterning by Plasma Stenciling
	<ol>
		<li>Shortly after plasma bonding pipette a 0.5 μl volume of PL or PO (100 μg/ml in 1x PBS) into the bottom center port. Within a matter of seconds the entire microfluidic circuit is filled by capillary action. Leave for a few minutes to fully coat the hydrophilic PDMS surfaces with the polyamines.</li>
		<li>Remove unbound polyamine molecules by aspiration-driven washing with 1x PBS (see Figure 2C) for 1 min.</li>
		<li>Remove all PBS from the ports and heat the device by illumination with a microscope. This increases evaporation from the ports for rapid water mask formation. The water mask (illustrated in Figure 3A and documented in Figure 3B) is established within ~5 min.</li>
		<li>Inspect the device by microscopy to ensure water masking is complete. Please note reservoir structures upstream of the array site are included in the mask design to prolong the stability of the water mask (~15 min).</li>
		<li>Insert stainless steel pins into the ports (Figure 2B). These are used to couple the plasma generated by an atmospheric pressure hand-held corona discharge or Tesla generator (e.g., operating at 2 MHz, 30 kV signal)19,20 into the vacant microfluidic channels. Point the tip of the plasma source close to the tip of one pin and plasma treat the microchannel for ≤1 sec. Use low light conditions to observe plasma patterning.</li>
		<li>Remove the water mask with a wash step. Use parallel aspiration from the upper 3 ports. Use 4 equal lengths of silicone tubing connected by a 4-way adaptor to an aspiration pump to achieve parallel aspiration. Start aspiration and dispense PBS into the bottom 3 ports to draw PBS through the microfluidic circuit. In this manner a PDMS-polyamine material contrast is produced (see Figure 3C). Evacuate PBS from the microfluidic circuit and leave for several hours to restore the native, hydrophobic state. This renders the PDMS fully non-permissive to cell adhesion.</li>
		<li>Should additional adhesion materials, such as laminin or fibronectin, be needed the following steps are required and should be done immediately after removing the water mask: Pipette a 10 μl volume of the graft co-polymer poly-L-lysine–poly(ethylene glycol) (PLL-g-PEG, 100 μg/ml, in PBS) into both bottom flanking channels, followed by aspiration from the upper flanking channels for 2 min.</li>
		<li>Without interrupting the flow, remove excess PLL-g-PEG by exchange with a PBS buffer. This selectively coats the non-polyamine-coated regions with the PEG moiety acting to prevent protein adsorption and cell adhesion17,21.</li>
		<li>Adhesion proteins such as laminin or fibronectin can then be co-localized with the poly-amine coating: Pipette 10 μl volumes of these ECM proteins (typically 10 μg/ml) into all three bottom ports. Aspirate from the top three ports until the device is filled with the protein solution and incubate for 1 hr to produce a good quality coating.</li>
		<li>Remove excess proteins by aspirating PBS from the bottom 3 ports.</li>
	</ol>
	</li>
	<li>Microfluidic Single Neuron Arraying
	<ol>
		<li>Prime the devices with appropriate media. Add 20 μl volumes to the bottom 3 ports and by parallel aspiration from the upper three ports rapidly fill the microfluidic circuit with media. Use a 4-way tubing connector to couple the 3 upper ports via equal-length silicone tubing to an aspiration pump.</li>
		<li>Add 20 μl of a disaggregated cell suspension (1 x 106 cells/ml) to each of the flanking inlet ports (0, in Figure 1).</li>
		<li>Equip an aspiration pump with a flow regulator or use a syringe for gentle aspiration from the upper 3 ports. Complete neuron arraying typically requires 1 min.</li>
		<li>Harvest excess neurons remaining in the 4 flanking ports with a pipette for arraying in subsequent microfluidic devices.</li>
		<li>Remove the tubing. Fill all ports with media and place the device in the incubator. Within a few hours the cells become fully adherent on the biomaterial pattern.</li>
	</ol>
	</li>
	<li>Neuron Culture, Selective Fluidic Treatment, and Immunostaining
	<ol>
		<li>Exchange the media by periodic perfusion using hydrostatic feed (a 2 mm column height difference is appropriately slow).</li>
		<li>Alternatively, submerge the microfluidic device in media, exchanging the media periodically (2–3 days) as with standard cell cultures.</li>
		<li>Selective fluidic treatments to either neuron culture compartment or the central neurite outgrowth compartment are achieved as follows: Dispense 20 μl of the test substance into the bottom port of the channel of interest, and aspirate from the port directly above. For lengthy treatments reduce the flow rate using a flow regulator and replenish the test substance as required.</li>
		<li>Immunostaining protocols vary for each molecular target and associated reagents. In order to counter the potential risk of shear-induced damage to the neurons a slow flow rate was used. This results in increased application times to fully perfuse the system, extending normal protocol times. Hydrostatic perfusion using ports with differing column heights (e.g. ,by 2 mm) was used to deliver reagents. More rapid reagent addition and removal using a pump can be used to reduce experimental timescales. Connect an aspiration pump to the upper 3 ports to draw reagents from the bottom 3 ports.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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'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 approach12–15, 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 (R2 &gt; R1) for transporting neurons to a linear array of microstructured apertures – 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 &gt; R2) to divert the streamlines into the serpentine path (0 → 2) to produce a bypass mode of operation for the removal of excess neurons.
<img alt="Neuron array chamber diagram with neurite outgrowth channels; microscopic image details." fo:content-width="5in" src="/files/ftp_upload/51389/51389fig1highres.jpg" width="500" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
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><tbody><tr><td>PDMS Sylgard 184</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>PDMS Elastosil RT 601</td><td>Wacker</td><td></td><td></td></tr><tr><td>Coverslips</td><td>VWR</td><td>630-1590</td><td>130-160 mm thick</td></tr><tr><td>3 mm biopsy punches</td><td>Kai Medical</td><td></td><td>Handle with care - extremely sharp</td></tr><tr><td>Tygon tubing</td><td>Fisher Scientific</td><td>S-50-HL</td><td>1.65 mm ID; 3.35 mm OD</td></tr><tr><td>1 ml syringe (Inkjekt and Omnifix)</td><td>Braun</td><td>6064204</td><td></td></tr><tr><td>4-Way tubing connector</td><td>VWR or Fisher Scientific</td><td></td><td></td></tr><tr><td>Flow regulator</td><td>Harvard Apparatus</td><td>722645</td><td></td></tr><tr><td>0.5 mm pins</td><td>Dressmaking Departments</td><td></td><td></td></tr><tr><td>PLL-g-PEG</td><td>SuSoS</td><td>PLL(20)-g[3.5]-PEG(5)</td><td>Stability in storage can be an issue</td></tr><tr><td>poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td></td></tr><tr><td>Polylysine-FITC</td><td>Sigma-Aldrich</td><td>P3069</td><td></td></tr><tr><td>Polyornithine</td><td>Sigma-Aldrich</td><td>P4957</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F2006</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>Inverted fluorescent microscope</td><td></td><td></td><td></td></tr><tr><td>Example aspiration pump</td><td>KNF Neuberger, Laboport</td><td>N811KVP</td><td></td></tr><tr><td>Hand held corona discharge device</td><td>Leybold-Heraeus, USA</td><td>VP23</td><td>May not comply with your country&#039;s safety standards</td></tr><tr><td>Femto plasma oven</td><td>Diener Electronic</td><td></td><td></td></tr><tr><td>Vacuum dessicator or centrifuge</td><td></td><td></td><td></td></tr></tbody></table>

preparation of neuronal co-cultures with single cell precision

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" alt="Young-Laplace equation; static equilibrium formula, pressure difference in fluid interface analysis." data-altupdatedat="1787630408000">, producing highly stable interfaces at microfluidic dimensions. The micropillars effectively anchor the water mask in place. Water mask formation is driven by evap...

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 ...

The overall goal of this procedure is to array single neurons in two microfluidic compartments for the preparation of a connected neuronal co-culture. This is accomplished by first replica molding the microfluidic device in PDMS and encapsulating the device by plasma bonding. The second step is to pattern polylysine within the microfluidic device.This is achieved by filling the device with polylysine, allowing a water mass to form by evaporation, and then using a plasma treatment to remove the exposed polylysine to create the biomaterial pattern. Next, use a syringe to aspirate the neurons through the microfluidic circuit for single neuron arraying. The device is placed in the tissue culture incubator for neurite outgrowths to extend and connect the two populations of neurons.The final step involves using an aspiration pump to selectively treat one compartment and monitor the propagation of materials and pathology within the neuronal circuit. Demonstrating the procedure will be Yayu Chang, and sarada of the ISIS lab. The main advantage of this technique over existing method, like commercially available PDMS component chamber, is that the neuron co culture are prepared with the single cell position for the analysis of individual neuro algorithms.This method can help answer key questions in the field of neuroscience. For example, what is the mechanism underlying the propagation of hyper phospho related tower aggregates in Alzheimer's disease or how to infect organism spreads throughout the brain? The technique is ideally suited to high throughput application since only a few hundred neurons are required for each experimental condition.Begin the procedure by mixing the PDMS pre polymer thoroughly with the curing agent at a ratio of 10 to one. Next, Degas the mixture in a vacuum desiccate for typically 20 minutes. Then place the two layered micro structured SU eight wafer on an 80 degree Celsius hot plate and align the polymer frames to each device on the wafer.Pour PDMS into the frame and allow one hour to ensure complete thermal curing. Once cured, remove the wafer from the hot plate and allow it to cool on a flat surface. Afterward, while wearing protective eyewear, use a rigid scalpel to gently prize the frame and PDMS from the wafer from one corner.Remove the PDMS mold from the frame and use a pair of scissors to trim the device. Apply the remaining PDMS to the entire wafer in order to protect it during storage. Upon peeling this PDMS layer off the wafer, any particulate contaminants are removed from the surface in readiness for repeat PDMS molding.Now prepare the six interface ports using a three millimeter biopsy punch with the micro structured features facing upwards to aid alignment of the ports with the microfluidic channels. Inspect the device for particulates and remove them using reversible adhesive tape. Next, prepare a thin PDMS layer mounted on a glass cover slip by pouring a small amount of the PDMS curing agent mixture onto the Petri dish.Press a cover slip onto the PDMS layer. Place it on the hot plate and thermally cure it for about 10 minutes. Shortly after cooling and while wearing safety glasses, use a scalpel to prize the cover slip.PDMS bilayer from the Petri dish to encapsulate the device. A good P-D-M-S-P-D-M-S bond is needed. Place the PDMS bilayer and the microfluidic device in the plasma oven for 40 seconds.Once plasma treated, press both parts together and allow to fully bond shortly after plasma bonding pipette 0.5 microliters of polylysine into the bottom center port. The entire microfluidic circuit should be filled by capillary action within seconds. Then leave it for a few minutes to fully coat the hydrophilic PDMS surfaces with polylysine.Next, remove the unbound polylysine by aspiration driven washing. Use a four-way union to connect the pump to the upper three ports with equal length tubing and start aspiration Add PBS to the bottom three ports and wash for one minute. Subsequently, detach the tubing and remove any remaining PBS from the ports.Transfer the device to a microscope and heat using illumination to increase evaporation from the ports for rapid water mask formation. Inspect the device by microscopy to ensure water masking is complete. Then insert stainless steel pins into the ports for coupling the plasma generated by a handheld corona discharge generator into the vacant microfluidic channels.Point the tip of the plasma source close to the tip of one pin and plasma. Treat the micro channel for one second. Afterward, remove the pins, connect the three upper ports to the pump.Start aspiration and dispense PBS into the bottom three ports. In order to draw PBS through the microfluidic circuit. Remove all PBS from the microfluidic circuit and leave overnight to complete biomaterial patterning.Here the pattern is visualized Using polylysine labeled with fluorescein. In this procedure, prime the device with appropriate media by adding 20 microliters to each of the bottom three ports. Then use parallel aspiration from the upper three ports to rapidly fill the microfluidic circuit with media.Add 20 microliters of a disaggregated cell suspension to each of the flanking bottom ports. Use a syringe to gently aspirate from the upper three ports. Complete neuron orang typically requires one minute.Then using a pipette harvest, excess neurons remaining in the ports for a ring in subsequent microfluidic devices. This example shows how neurons are arrayed with single cell precision. After that, submerge the device in media and place the device in the incubator for the cells to become fully adherent on the biomaterial pattern within a few hours.As with normal cultures, the media should be replenished periodically to selectively treat either neuron culture compartment or the central neuro outgrowth compartment. Dispense 20 microliters of the test substance into the bottom port, connected to the channel of interest and aspirate from the port directly above. In this example, a selective treatment has been applied to the central compartment, and in this example, the flanking compartment has been treated.Treatments were established in one minute and were maintained for 60 minutes. The neuron co cultures can then be monitored to measure the propagation of materials or pathology. Once mastered, many microfluidic devices can be prepared in a single morning.After watching this video, you should have a good idea of how to prepare the minimalistic neuronal cultures in your own labs and use these to investigate mechanisms underlying material and disease propagation in the brain.

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

Research

JoVE Journal

Neuroscience

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Preparation of Dissociated Mouse Cortical Neuron Cultures

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video demonstrates a method to differentiate human neural progenitor cells (NPCs) into neurons. The NPCs are introduced onto an established mouse astrocyte-rat cortical neuron co-culture, promoting NPC differentiation into neurons. The differentiation is confirmed by visualizing the cells under a confocal microscope.

A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons

This video illustrates a method for establishing an indirect neuron-astrocyte co-culture using a compartmentalized approach. In this setup, neurons and astrocytes are spatially separated but share a conditioned medium. Neurotrophic factors released by astrocytes promote neuron differentiation and the formation of synaptic connections, suggesting neuron-glia interactions.

An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Learn to prepare interconnected neuronal co-cultures with single cell precision using microfluidic arraying and plasma patterning techniques. This method enables efficient neuroscience research with minimal cell inputs.

Preparation of Neuronal Co-cultures with Single Cell Precision

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