Name: Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures (Video) | JoVE Journal
Uploaded: 2008-10-17T00:00:00.000+00:00
Duration: 7 min 43 s
Description: Learn to prepare compartmented cultures for dorsal root ganglia neurons using E15 rat embryo cells. This method enables axonal growth studies and neurotrophin signaling research in controlled environments.

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5 minutes

We would like to thank Katharina Cosker and Stephanie Courchesne for helpful discussions.

Preparation of reagents
<ol>
<li>Collagen coating: collagen coat p35 tissue culture plates and place in an oven at 37°C for 2 days before greasing the dividers. The final concentration of collagen should be at .71 mg/ml diluted in .001N HCl.  Then, add 1 ml of mixture per plate.</li>
<li>Grease loaders: In order to fill the grease loader, a 60mL syringe must first be filled with Corning vacuum grease. Use the syringe to fill the grease loader, wrap it in foil and then autoclave for 45 minutes.</li>
<li>Teflon dividers: the dividers can be re-used after each experiment but must first be properly cleaned. Remove the divider from the plate, wipe off all of the remaining grease and place in sulfuric acid for 2 days. After removing from the acid, rinse with water 3X, boil for 20 minutes, allow to dry, place in a glass p100 petri dish and autoclave for 20 minutes.</li>
<li>N2-methylcellulose: Weigh out 1.5g of methylcellulose and place it in a 500mL bottle. Add a stir bar and autoclave it for 20 minutes on dry (from this point all work must be sterile). Next, add 500 mLs of serum free media (N2), and stir in a cold room until it dissolves. Aliquot into 50 mL conicals and freeze at -20°C.  For working stock, aliquot one of the 50 mL conicals into 1mL tubes and freeze at -20°C.</li>
<li>DRG media: DMEM, 5% Heat inactivated horse serum, and 1% penicillin streptomycin.</li>
<li>100ng/mL DRGN media: The stock concentration of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) is 1mg/mL. Dilute each of the neurotrophins 1:10,000 into the DRG media. Cultures can be grown in NGF alone; this alters the complement of neurons that survive in the cultures. Note: When needed, the concentration of (cytosine arabinoside) AraC is 1uM and used at a final concentration of 0.3uM. This will inhibit growth of Schwann cells and other glia. </li>
<li>10ng/mL DRGN media:  Dilute the 100ng/mL DRGN media (1:10) with DRG media. <img src="/files/ftp_upload/old/951_2008_10_27_6/951_Figure-01.png" alt="Archery fletching tool and adhesive setup for attaching vanes to arrow shafts, showing equipment." width="392" height="294" data-altupdatedat="1761735611000" data-alt="Figure 1"> Figure 1. Tools needed for set-up </li>
</ol>
Setting up the compartmented chambers (start this process 1-2 days before the dissection)
<ol>
<li>Make a scratch in the middle of a collagen coated p35 dish with an outward motion.</li>
<li>Place 30ul of N2-methylcellulose on the middle of the scratch. Set dishes aside until divider is greased.</li>
<li>Attach a 23-gauge luer stub adapter to the grease loader. Grip the Teflon divider with a pair of 90° angle hemostats and lay it flat with the divider facing up under a microscope. Trace the divider with grease making sure that each time the adapter is placed at a new starting point the adapter is inserted into the grease from the previous step so that there is a continuous line of grease (see diagram). Once the grease is applied to the entire divider, turn one of the prepared p35 dishes upside down and place it so the N2-methylcellulose is over the middle compartment. Press down on the bottom of the dish with a pair of tweezers. Make sure to press on the inside of the divider in four corners (upper left, lower right, upper right, lower left, indicated in diagram by "X"). <img src="/files/ftp_upload/old/951_2008_10_9_2/951_Figure-02.png" alt="Chemotaxis assay diagram, illustrating organism movement toward chemical stimulus, with grease barrier." width="171" height="183" data-altupdatedat="1761735611000" data-alt="Figure 2"> Figure 2: Steps to greasing the divider Note: It is important to press firmly enough so that the grease makes a complete seal with the dish, but if too much pressure is added, the axons will not cross into the side compartments.  Pick up the hemostats, turn it over and unclamp the divider. Lastly, place the dish with the divider firmly attached under the microscope focusing on the bottom of the middle compartment. With the grease loader, make a small barrier (.25cm) so that once the cells are placed in the middle compartment they cannot leak out. </li>
<li>After having set up several cultures, place DRG media in each of the side compartments and place in an incubator in which the cells will be maintained.  Allow the cultures to sit for several hours and then check for leakage. If the media has leaked into the middle compartment, then the culture is unusable. Note: When first learning this technique, it is important to set up more cultures than are needed for an experiment, as several will be leaky. <img src="/files/ftp_upload/old/951_2008_10_9_3/951_Figure-03.png" alt="Static equilibrium, valve comparison diagram; illustrating proper seal versus leakage issues." width="254" height="191" data-altupdatedat="1761735611000" data-alt="Figure 3"> Figure 3: "Good vs leaky" culture </li>
</ol>
Maintaining DRG neurons in compartmented cultures
<ol>
<li>Day 1:  Replace DRG media in the side compartments with 100ng/mL DRGN media + AraC.  Perform dissection and add cells to center compartment (100,000 cells).  </li>
<li>Day 2:  Add 10ng/mL media + AraC to the outside of the Teflon divider until the media flows over the grease barrier and exchanges fluid with the center compartment.</li>
<li>Day 3:  Replace media in the side compartments to 100ng/mL DRGN omitting the AraC and the surround with 10ng/mL DRGN omitting the AraC.</li>
<li>Day 6:  Replace media in the side compartments to 1ng/mL + AraC and the surround with DRG media + AraC.</li>
<li>Day 9:  Use for experimentation. <img src="/files/ftp_upload/old/951_2008_10_9_5/951_Figure-04.png" alt="Neuronal imaging; fluorescence microscopy, cell bodies, and distal axons; comparative diagram." width="267" height="148" data-altupdatedat="1761735611000" data-alt="Figure 4"> Figure 4: IHC images of cell bodies and distal axons Note:  When changing the media, it is important to aspirate the liquid from the top of the each side compartment. Also, never change the media from the middle compartment itself, only from the surround, and let it flow over the grease barrier into the center. </li>
</ol>

<ol><li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Independent+Control+of+the+Local+Environment+of+Somas+and+Neurites%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Independent Control of the Local Environment of Somas and Neurites.</a> Methods in Enzymology. 58, 302-307 (1979).</li>
<li>Watson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotrophins+use+the+Erk5+pathway+to+mediate+a+retrograde+survival+response%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Neurotrophins use the Erk5 pathway to mediate a retrograde survival response.</a> Nature Neuroscience. 4, 981-988 (2001).</li>
<li>Heerssen, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynein+motors+transport+activated+Trks+to+promote+survival+of+target-dependent+neurons%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Dynein motors transport activated Trks to promote survival of target-dependent neurons.</a> Nature Neuroscience. 7, 596-603 (2004).</li>
<li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microfluidic+culture+platform+for+CNS+axonal+injury%2C+regeneration+and+transport%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2, 599-605 (2006).</li>
<li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+culture+platform+for+neuroscience+research%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 4, 2128-2136 (2006).</li>
<li>Ure, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retrograde+transport+and+steady-state+distribution+of+125I-nerve+growth+factor+in+rat+sympathetic+neurons+in+compartmented+cultures%5BTitle%5D)+AND+%22J+Neuroscience%22%5BJournal%5D)">Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures.</a> J Neuroscience. 4, 1282-1290 (1997).</li>
</ol>

In this video, we have demonstrated how to prepare and maintain compartmented chambers for use in culturing DRG neurons. Done properly, this system allows separation of the cell body from the axon in order to study mechanisms by which neurotrophins signal across long axons. Since there is fluidic isolation between the compartments, it allows for selective stimulation or treatment of one compartment without the other compartments being affected. Compartmented chamber cultures can support other cell types including sympath...

<table><tbody><tr><td>collagen</td><td>Reagent</td><td>BD Biosciences</td><td>354249</td><td></td></tr><tr><td>N2-methylcellulose 400CPS</td><td>Reagent</td><td>Sel-Win Chemicals</td><td></td><td></td></tr><tr><td>Teflon divider</td><td>Other</td><td>Tyler Research</td><td>CAMP10</td><td>many other types of dividers are available</td></tr><tr><td>Pin rake</td><td>Tool</td><td>Tyler Research</td><td>Camp-PR</td><td></td></tr><tr><td>Grease loader</td><td>Tool</td><td>Tyler Research</td><td>Camp-GLSS</td><td></td></tr><tr><td>DMEM</td><td>Reagent</td><td>Fisher Scientific</td><td>MT10017CV</td><td></td></tr><tr><td>NGF</td><td>Reagent</td><td>PeproTech Inc</td><td>450-01</td><td></td></tr><tr><td>BDNF</td><td>Reagent</td><td>PeproTech Inc</td><td>450-02</td><td></td></tr><tr><td>High vacuum grease</td><td>Reagent</td><td>Fisher Scientific</td><td>14-635-5D</td><td></td></tr><tr><td>AraC</td><td>Reagent</td><td>Sigma-Aldrich</td><td>C-1768</td><td></td></tr><tr><td>23 gauge luer stub adapter</td><td>Tool</td><td>Fisher Scientific</td><td>427565</td><td></td></tr><tr><td>90&amp;#x00B0; angle hemostats</td><td>Tool</td><td>Roboz Surgical Instruments Co.</td><td>RS-7035</td><td></td></tr></tbody></table>

preparation and maintenance of dorsal root ganglia neurons in compartmented cultures

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

This procedure begins with making a scratch with a pin rake onto the center of a collagen coated plate, and pipetting 30 microliters of Methylcellulose onto the center of the scratches. Grease each divider using a grease loader and attach it to a collagen coated plate plate. 100, 000 cells from E 15 rat embryos into the middle compartment.After nine days, the cells will have extended axons into the side compartment and will be ready for experimentation. Hi, I'm Maria Murphy from the laboratory of Dr.Rosalyn Siegel in the Department of Pediatric Oncology at the Dana-Farber Cancer Institute. Today we will show you a procedure for the preparation and maintenance of Dosa root ganglia neurons in compartmented cultures.We use this procedure on our laboratory to study neurotrophin signaling. So let's get started. Before setting up the compartmented chambers, P 35 tissue culture plates must be coated with collagen.To do this, add one milliliter of a 0.71 milligram per milliliter solution of collagen in 0.001 normal HCL to the plates and incubate them at 37 degrees Celsius for two days. After two days, make a scratch in the middle of the collagen coated plates with a pin rake. Use an outward motion.Then place 30 microliters of the N two Methylcellulose on the middle of the scratch. Set the dishes aside. Next, attach a 23 gauge lure stub adapter to the grease loader.Grip the Teflon divider with a pair of 90 degree angle hemostats and lay it flat with the divider facing up under a microscope. Looking through the scope, race the divider with grease. Make sure that each time the adapter is placed at a new starting point, it is inserted into the grease from the previous step so that there is a continuous line of grease.Once the grease is applied to the entire divider, turn one of the prepared P 35 dishes upside down and place it so that the N two Methylcellulose is over the middle compartment. Press down on the bottom of the dish with a pair of tweezers. Make sure to press on the inside of the divider in four corners.It is important to press firmly enough so that the grease makes a complete seal with a dish. But if too much pressure is added, the axons will not cross into the side compartments. Next, pick up Hemostats.Turn over the dish and unclamp the divider. Then place the dish with the divider firmly attached under the microscope, focusing on the bottom of the middle compartment. Use the grease loader to make a small barrier connecting both sides of the divider about a quarter of a centimeter high.This step will prevent the cells from leaking out once they're placed in the middle compartment. After setting up several cultures, place DRG media in each of the side compartments. Then place in the incubator where the cells will be maintained.Allow the cultures to sit for several hours and then check for leakage. If the media has leaked into the middle compartment, then the culture is unusable. When first learning this technique, it is important to set up more cultures than are needed for an experiment, as several will probably be leaky.After confirming that the compartmented chambers are usable, proceed with the maintenance of the dorsal root ganglia neuron culture. Prior to experimentation, the DRG neurons are maintained in the compartmented cultures. Over a period of nine days at 37 degrees Celsius on day one, replace the DRG media in the side compartments with 100 nanograms per milliliter.DRGN media plus A-C-D-R-G-N media contains both nerve growth factor and brain derived neurotrophic factor. The recipe for this media is available in our written protocol. Perform the dissection on E 15 rat embryos and add 100, 000 cells to the center compartment.On day two. Add 10 nanograms per milliliter DRGN media plus a C to the outside of the Teflon divider until the media flows over the grease barrier and exchanges fluid with the center compartment on day three, replace media in the side compartments with 100 nanogram per milliliter. DRGN omitting the a c.Also replace the surround with 10 nanogram per milliliter. DRGN omitting the RSC on day six. Replace media in the side compartments to one nanogram per milliliter, DRGN plus RSC and the surround with DRG media plus RSC.On day nine, the compartmented culture is ready for experimentation. After experimentation is completed, the Teflon dividers can be reused, but must be properly cleaned. To do this, remove the divider from the plate and wipe off all of the remaining grease.Then place in sulfuric acid for two days. After two days, remove dividers from the acid and rinse them with water three times, then boil for 20 minutes and allow to dry. Place the dry dividers in a glass P 100 Petri dish and autoclave for 20 minutes after sterilization, they'll be ready for future experiments.This compartmented culture system allows separation of the cell body from the axon in order to study mechanisms by which neurotrophins signal across long axons. Since there is fluidic isolation between the compartments, it allows for selective stimulation or treatment of one compartment without the other compartments being affected. We've just shown you how to prepare and maintain dosa root ganglia neurons in compartment to cultures.When doing this procedure, it's important to remember to press gently when sealing the grease divider to the collagen coated plate. Aspirate the liquid from the top of each side compartment when changing the media as to not disturb the axons. Never change the media directly from the middle compartment when feeding the neurons.Change only the media from the surround and let it flow over the grease barrier into the middle compartment. So that's it. Thanks for watching and good luck with the experiments.

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Research

JoVE Journal

Biology

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

This video demonstrates a procedure for isolating dorsal root ganglion (DRG) neurons from rat embryos and co-culturing them with Schwann cell precursors to generate mature Schwann cells. The isolated DRGs are processed to isolate neurons and glial cells. The isolated cells are cultured to purify the neurons, which are then co-cultured with Schwann cell-like cells (SCLCs) in a co-culture medium. Interaction with the DRG neurons leads to the maturation of SCLCs into Schwann cells.

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A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures

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Co-culturing of a Dorsal Root Ganglion Explant with Schwann Cells for Neuron Myelination

This video demonstrates a technique for isolating and culturing dorsal root ganglia (DRG) cells from the rat spinal column. DRGs excised from the spinal column are treated with enzymes and mechanically dissociated to obtain a single-cell suspension. The cells are cultured in a medium containing inhibitors and growth factors that limit glial cell proliferation and promote neuronal growth, respectively.

Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

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Establishing In Vitro Models of Dorsal Root Ganglia Culture: Complementary Approaches for Investigating Cancer-Nerve Crosstalk

Learn to prepare compartmented cultures for dorsal root ganglia neurons using E15 rat embryo cells. This method enables axonal growth studies and neurotrophin signaling research in controlled environments.

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

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