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 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 &#176;C hotplate and align polymer frames to each device on the wafer. Pour PDMS into the frames to a depth of &#8805;5 mm and allow &#62;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&#8211;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="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51389/51389fig2highres.jpg" width="500" /> 
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://jove.unicartagenaproxy.elogim.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 &#8804;5&#176;). 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="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51389/51389fig3highres.jpg" width="500" /> 
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&#8211;TRITC to a plasma-patterned PL-FITC coating. This was used for the registration of&#160;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://jove.unicartagenaproxy.elogim.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 &#956;l&#160;volume of PL or PO (100&#160;&#956;g/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.,&#160;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 &#8804;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&#160;&#956;l volume of the graft co-polymer poly-L-lysine&#8211;poly(ethylene glycol) (PLL-g-PEG, 100&#160;&#956;g/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&#160;&#956;l volumes of these ECM proteins (typically 10&#160;&#956;g/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&#160;&#956;l 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&#160;&#956;l 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&#8211;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&#160;&#956;l 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&#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://jove.unicartagenaproxy.elogim.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

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...

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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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13.6K Views.  ISAS. 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.

Video: Preparation of Neuronal Co-cultures with Single Cell Precision

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

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

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.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Developmental Biology

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

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.

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Biology

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

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.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (&#947;-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. 
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. 
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival. 
Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed material.

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

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.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

Primary Neuronal Cultures

The complexity of the brain often requires neuroscientists to use a simpler system for experimental manipulations and observations. One powerful approach is to generate a primary culture by dissecting nervous system tissue, dissociating it into single cells, and growing those cells in vitro. Primary cultures make neurons and glia easily accessible to the experimental tools required for techniques like genetic manipulation and time-lapse imaging. Furthermore, these cultures represent a highly controllable environment in which to study complex phenomena such as cell-cell interactions.
This video provides an overview of the major steps in producing primary neuronal cultures, which include selecting and dissecting the tissue of interest, mechanically and chemically breaking down the tissue to produce a single cell suspension, plating the cells, and maintaining the cultures in the appropriate media. Several example experiments are also presented to show how cultured cells can be used to investigate protein trafficking, morphological changes, and electrophysiology in living neurons.

Primary Neuronal Cultures and Electrophysiology

The complexity of the brain often requires neuroscientists to use a simpler system for experimental manipulations and observations. One powerful approach ...

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Advanced Biology

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

Source: Su, C. T. E., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52408">An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System.</a> J. Vis. Exp. (2015).
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.

Technika kohodowli do różnicowania ludzkich neuronalnych komórek progenitorowych w neurony

1. Generation of Induced Pluripotent Stem Cells

	Culture human fibroblasts in DMEM supplemented with 10% fetal bovine serum (FBS) in 6-well plates.
	...

Preparation of Neuronal Co-cultures with Single Cell Precision

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Rozdziały w tym wideo

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

Preparation of Dissociated Mouse Cortical Neuron Cultures

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABA_A Receptors

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Primary Neuronal Cultures

Technika kohodowli do różnicowania ludzkich neuronalnych komórek progenitorowych w neurony