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 JoVE Biology

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

1,2, 1,2

1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School

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    Summary

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

    Date Published: 10/17/2008, Issue 20; doi: 10.3791/951

    Cite this Article

    F. Pazyra-Murphy, M., A. Segal, R. Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures. J. Vis. Exp. (20), e951, doi:10.3791/951 (2008).

    Abstract

    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.

    Protocol

    Preparation of reagents

    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. DRG media: DMEM, 5% Heat inactivated horse serum, and 1% penicillin streptomycin.
    6. 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.

    7. 10ng/mL DRGN media:  Dilute the 100ng/mL DRGN media (1:10) with DRG media.

      Figure 1


      Figure 1. Tools needed for set-up

    Setting up the compartmented chambers (start this process 1-2 days before the dissection)

    1. Make a scratch in the middle of a collagen coated p35 dish with an outward motion.
    2. Place 30ul of N2-methylcellulose on the middle of the scratch. Set dishes aside until divider is greased.
    3. 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").

      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.
    4. 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.

      Figure 3

      Figure 3: "Good vs leaky" culture

    Maintaining DRG neurons in compartmented cultures

    1. 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). 
    2. 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.
    3. 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.
    4. Day 6:  Replace media in the side compartments to 1ng/mL + AraC and the surround with DRG media + AraC.
    5. Day 9:  Use for experimentation.


      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.

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    Discussion

    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 sympathetic neurons from the superior cervical ganglia, retinal ganglion neurons, and cortical neurons. Spatial understanding of neurotrophin signal transduction may provide novel insights into treatments of neurodegenerative disorders. Several neurodegenerative disorders, including Alzheimer's disease, Huntington's disease and motor neuron disease, are associated with defects in axonal transport. Recent studies have used microfluidic chambers instead of these compartmented chambers. The microfluidic chambers4,5 have several advantages for imaging analysis.

    Prior studies have tested the ability of these cultures to prevent diffusion between the axon and the cell body compartment1,3,6. This can easily be tested by adding low concentrations of a dye such as trypan blue to one compartment only, and look for diffusion of the dye. There should be little or no diffusion visible within 24 hours.

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    Disclosures

    Acknowledgements

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

    Materials

    Name Type Company Catalog Number Comments
    collagen Reagent BD Biosciences 354249
    N2-methylcellulose 400CPS Reagent Sel-Win Chemicals
    Teflon divider Other Tyler Research CAMP10 many other types of dividers are available
    Pin rake Tool Tyler Research Camp-PR
    Grease loader Tool Tyler Research Camp-GLSS
    DMEM Reagent Fisher Scientific MT10017CV
    NGF Reagent PeproTech Inc 450-01
    BDNF Reagent PeproTech Inc 450-02
    High vacuum grease Reagent Fisher Scientific 14-635-5D
    AraC Reagent Sigma-Aldrich C-1768
    23 gauge luer stub adapter Tool Fisher Scientific 427565
    90° angle hemostats Tool Roboz Surgical Instruments Co. RS-7035

    References

    1. Campenot, RB. Independent Control of the Local Environment of Somas and Neurites. Methods in Enzymology 58 302-7, (1979).

    2. Watson, FL., et al. Neurotrophins use the Erk5 pathway to mediate a retrograde survival response. Nature Neuroscience 4 981-88, (2001).

    3. Heerssen, HM., et al. Dynein motors transport activated Trks to promote survival of target-dependent neurons. Nature Neuroscience 7 596-603, (2004).

    4. Taylor, AM., et al. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nature Methods 2 599-605, (2005).

    5. Park JW., et al. Microfluidic culture platform for neuroscience research. Nat Protoc. 4 2128-36, (2006).

    6. Ure DR., et al. Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures. J Neuroscience 4 1282-90, (1997)

    Comments

    43 Comments

    Hi I have just watch your presentaion on the Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures and it was very impressive. I am intersted in the function of the methylcellulose, dŒs it provide a space through which the axons can pass, do the cell bodies settle on the collagen surface. Regards Paul
    Reply

    Posted by: AnonymousNovember 19, 2008, 7:27 AM

    Hi Paul-
    Thank you for your comment.  The cell bodies do settle onto the collagen surface.  As for the small amount of N²-methylcellulose that is placed on the center of the scratch, the media acts as a space for the axons to extend easily on and the methylcellulose is used to thicken the media slightly. 

    Good Luck,
    Maria
    Reply

    Posted by: AnonymousDecember 15, 2008, 3:06 PM

    Hi, your method looks simple and powerfull. It's possible to make an immuno staining without to remove the teflon divider? DŒs the divider cause troubles during immunofluorescence staining for example? Regards Giuseppe 
    Reply

    Posted by: AnonymousDecember 13, 2008, 7:35 AM

    Hi Giuseppe-
    Thank you for your comment. Fix, wash and add antibodies as usual within the compartments for immunostaining. Remove the divider, carefully wipe away excess grease and coverslip before imaging.

    Good Luck,
    Maria
    Reply

    Posted by: AnonymousDecember 15, 2008, 3:52 PM

    Hi, beautiful work, congratulations! Have you tried to cultivate adult DRG also? Best regards and good luck! Otilia
    Reply

    Posted by: AnonymousFebruary 5, 2009, 6:34 AM

    Hi Otilia- Thank you for your comment.  We have not tried cultivating adult DRGs in this system.   Good Luck, Maria
    Reply

    Posted by: AnonymousMarch 19, 2009, 12:03 PM

    Dear Otilia,

    I too was really impressed by Maria's work and wanted to use the technique for my work. I just wanted to let you know that I have been using adult DRGs in this system and it still works brilliantly.

    Best regards,

    Philippa
    Reply

    Posted by: AnonymousSeptember 2, 2009, 1:02 PM

    Hi, I hvae not been able to watch the video.  Could you please email me  a copy. Sincerely, Supinder Bedi, Ph.D. University of Texas, Houston
    Reply

    Posted by: AnonymousFebruary 11, 2009, 3:39 PM

    Hi, We are very interested in your method, that’s great!
    We have one question: on day1, how do you "perform dissection and add cells to center compartment (100,000cells)"?  Can you please provide more details? Best regards Yi
    Reply

    Posted by: AnonymousMarch 5, 2009, 3:52 PM

    Hi Amazing video; really impressive and well constructed. I'm hoping to use the compartmented culture system for some of my experiments and so am currently trying to set it up however as i hope to do immunostaining of the cultures I was just wondering whether you use just normal polystyrene cell culture dishes or glass-bottomed dishes/ dishes with a glass coverlip plated in it? If you use glass what thickness and type of glass do you use? The reason I ask is I've been seeing whether I can coat glass coverslips with collagen in order to be able to detach them from the dishes and mount them onto microscope slides later however I don't seem to be able to coat the borosilicate glass coverslips that I have. I've tried all sorts of methods including your protocol here but it seems that the collagen dŒsn't want to stick to the glass. They are definitely degreased throughly therefore I was wondering whether it was an issue with the charge of the glass and whether there was a different sort of glass I should be using. Any suggestions would be really appreciated. Best regards, Philippa
    Reply

    Posted by: Philippa M.March 19, 2009, 7:25 AM

    Hi Philippa- Thank you for your comment.  We only use polystyrene cell culture dishes that have been coated with collagen.  It is impossible to make the necessary scratches on the glass.  We often do immunostaining and image with this system, however, there is another system, microfluidic chambers, that may give you higher quality images. Good Luck, Maria  
    Reply

    Posted by: AnonymousMarch 19, 2009, 11:56 AM

    Dear Philippa, I have seen the video and read your comment only yesterday, but maybe this can help: at www.mattek.com, you can order 35mm or 50mm dishes with a partially glass bottom (coverslip that you can even take out afterwards and that is either uncoated or coated with collagen or poly-D-lysine). We have been using them on a regular basis for live cell imaging and they're very useful. From my side, I was wondering if you made any progress with scratching glass dishes, because I would like to do this for my particular experiment. Best regards, Katrien
    Reply

    Posted by: Katrien J.August 28, 2009, 5:01 AM

    Hi Amazing video; really impressive and well constructed. I'm hoping to use the compartmented culture system for some of my experiments and so am currently trying to set it up however as i hope to do immunostaining of the cultures I was just wondering whether you use just normal polystyrene cell culture dishes or glass-bottomed dishes/ dishes with a glass coverlip plated in it? If you use glass what thickness and type of glass do you use? The reason I ask is I've been seeing whether I can coat glass coverslips with collagen in order to be able to detach them from the dishes and mount them onto microscope slides later however I don't seem to be able to coat the borosilicate glass coverslips that I have. I've tried all sorts of methods including your protocol here but it seems that the collagen dŒsn't want to stick to the glass. They are definitely degreased throughly therefore I was wondering whether it was an issue with the charge of the glass and whether there was a different sort of glass I should be using. Any suggestions would be really appreciated. Best regards, Philippa
    Reply

    Posted by: Philippa M.March 19, 2009, 8:12 AM

    Hi Amazing video; really impressive and well constructed. I'm hoping to use the compartmented culture system for some of my experiments and so am currently trying to set it up however as i hope to do immunostaining of the cultures I was just wondering whether you use just normal polystyrene cell culture dishes or glass-bottomed dishes/ dishes with a glass coverlip plated in it? If you use glass what thickness and type of glass do you use? The reason I ask is I've been seeing whether I can coat glass coverslips with collagen in order to be able to detach them from the dishes and mount them onto microscope slides later however I don't seem to be able to coat the borosilicate glass coverslips that I have. I've tried all sorts of methods including your protocol here but it seems that the collagen dŒsn't want to stick to the glass. They are definitely degreased throughly therefore I was wondering whether it was an issue with the charge of the glass and whether there was a different sort of glass I should be using. Any suggestions would be really appreciated. Best regards, Philippa
    Reply

    Posted by: Philippa M.March 19, 2009, 2:04 PM

    Hi,   Could you please elaborate a bit more on the DRG dissection and how you get to 100,000 cells please Thanks,   Gustavo Ayala R. Clarence and Irene H Fullbright Chair in Pathology Professor Baylor College of Medcine  
    Reply

    Posted by: AnonymousMarch 24, 2009, 4:58 PM

    hi, thank you for the presentation, your novel model for preparation of drg neurons seem to be very efficient. can you please elaborate how did you get the neurons and how old was the rat fetus? thanks ahead, Amit Moran, moranamit@gmail.com    
    Reply

    Posted by: AnonymousMarch 30, 2009, 11:58 PM

    It is very interesting. I am wondering regarding the use of NGF. You have used Recombinant human beta NGF. Is there any special reason you used human NGF? or is it ok that we can use rat NGF?
    Reply

    Posted by: AnonymousJune 23, 2009, 3:16 PM

    Hi Anand-

    We prefer to use the recombinant human NGF but it is certainly okay to use rat NGF.

    Good Luck,
    Maria
    Reply

    Posted by: AnonymousJune 30, 2009, 12:51 PM

    Thanks for your presentation. Have you ever tried cultivating hippocampal neurons? If it 's, what did you coated with your dish, collagen or poly-l-lysine?

    Regards


    Mei
    Reply

    Posted by: AnonymousAugust 18, 2009, 4:35 PM

    Hi Mei-

    Sorry, we have never cultivated hippocampal neurons using this system. There is a paper, Ivins et al., 1998, that uses a modified compartmented culture system that you may find helpful.

    Good Luck,
    Maria
    Reply

    Posted by: AnonymousAugust 21, 2009, 1:39 PM

    Dear colleges, thank you very much for nice performance and demonstration of this method!
    I have a question. How do you think could I use this compartmented culture to investigated axonal degeneration. If I will add a substance under the investigation to axonal part of chamber, how could I be sure that this compound dŒs not penetrate to cell body part?
    Thank you very much for answer beforehand and good luck in your future experiment!!!
    Reply

    Posted by: Liudmila E.September 28, 2009, 9:17 AM

    Hi Lula-
    If set up properly (no leakage) these chambers are fluidically isolated.

    Good Luck,
    Maria
    Reply

    Posted by: AnonymousJanuary 13, 2010, 2:54 PM

    Hi Maria,
    I will like to confirm the concentration of collagen coating that you use have on the protocol (0,71mg/mL diluted in 0,001N HCl). In our lab we use collagen coating for cultures and the concentration is very less.
    Regards
    AleMorán
    Reply

    Posted by: AnonymousSeptember 28, 2009, 1:09 PM

    Hi Alemor-
    Yes, that is the correct concentration of collagen.
    Good Luck,
    Maria
    Reply

    Posted by: AnonymousJanuary 13, 2010, 2:39 PM

    Hi, It is quite interesting. I need some help in isolating DRG. I tried. But not able to identify them. Can you pls help me.
    Thanks in advance.
    Reply

    Posted by: AnonymousOctober 26, 2009, 3:44 PM

    Hi Anand-
    The DRGs are located along each side of the spinal cord. In the E15 rats, the DRGs are clearly visible as ganglia along the spinal cord. At older ages the DRGs are encased within the vertebrae.
    Good Luck,
    Maria
    Reply

    Posted by: AnonymousJanuary 13, 2010, 2:42 PM

    Hi,
    Great presentation!
    I was wondering, what antibodies did you use for the IHC images?

    Thanks,
    Amy
    Reply

    Posted by: Amy M.December 1, 2009, 8:43 PM

    Hi,

    many thanks for posting this really helpful video. When you apply the grease on the divider do you work under a hood? If not, how do you maintain sterility? I have tried working outside a hood and I have had problem of contamination (all solutions, and tools have been either filtered or autoclaved)

    Kind regards,

    Ale
    Reply

    Posted by: AnonymousJanuary 13, 2010, 11:31 AM

    Hi Ale-
    It would be best if you set up the cultures in a dissecting hood if you are having problems with contamination. We use UV to sterilize the area but the hood that we work under has no air flow and is therefore not a completely sterile environment.

    Good Luck,
    Maria
    Reply

    Posted by: AnonymousJanuary 13, 2010, 2:51 PM

    Hi
    Beautiful work! And I have been tried to constructed the compartmented cultures for many times but failed .I don't know why. I wonder how do you get to 100,000 cells .Could you please elaborate a bit more on the DRG dissection .In my compartmented cultures ,there are only a few axons across the barrier and get to the distal compartment.I have tried it for many times but the results are the same.I guss if the cell differentiation is not good enough or something wrong with the applying grease.I have no idea .So i think if you can give me your email and I have many problems to ask you.My email is jjdongch@gmail .com. I am looking forward to your reply .Thank you!
    Reply

    Posted by: jingjing d.May 17, 2012, 7:39 AM

    Hi!, excellent video!. We are reproducing this system, but now, we have problem with collagen coating. It does not gelled 3 or 5 days after!, in fact, it does not change. We´re using the same collagen (BD Bioscience ,354249) and your final concentration (0.71mg/ml diluted in 0.001N HCl). What recommendations do you have?.

    Kind Regards!
    Reply

    Posted by: DIANA M.January 9, 2014, 1:00 PM

    Hi Diana-
    The plates need to be put in a dry oven at 37 degrees for 3 days. Are you doing that? Also, the collagen doesn't gel, it dries completely. Good luck and feel free to email me with any additional questions. maria_pazyra@dfci.harvard.edu
    Reply

    Posted by: AnonymousJanuary 9, 2014, 1:29 PM

    Great article. What are the product numbers and companies for methylcellulose and N2 serum free media?
    Reply

    Posted by: Eric W.June 17, 2014, 3:04 PM

    Hi Eric- The methylcellulose we currently use is from Xenex (catalog # E4M) and the serum free media is just plain DMEM. Good Luck, Maria
    Reply

    Posted by: AnonymousJune 17, 2014, 3:21 PM

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