This work was supported by an NSF Grant (2152065) and an NSF CAREER Award (1846857). The authors would like to thank all current and past members of the Wachs Lab for contributing to this protocol. Diagrams in Figure 1 were made in Biorender.

DRG harvest was performed in compliance with the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska-Lincoln. Female Sprague Dawley rats aged 12 weeks (~250 g) were used for the study. The details of the animals, reagents, and equipment used in the study are listed in the Table of Materials.
1. Multi-compartment device fabrication and assembly
<ol>
	<li>Computer-aided design and 3D printing of the multi-compartment device 
	NOTE: The MC device consists of three compartments for the isolation of DRG cell bodies and neurites (Figure 2A). The MC device is not commercially available; however, the STL file is provided herein (Supplementary Coding File 1) to enable 3D printing. Printing the MC takes one (1) day to complete.</li>
	<li>Upload the STL file of the MC to a Resin 3D printer using compatible software. 
	NOTE: The material ideal for printing is a high-temperature resin. A key advantage of using high-temperature resin is it is biocompatible and can be autoclaved and reused. A Digital light processing (DLP) 3D printer or other 3D printers could also be used to print the device.</li>
	<li>Transfer the printed devices into a washing solution for 30 min in isopropanol (&#62;96%) to remove residual resin from the surface of the printed MC device.</li>
	<li>Post-cure the devices at 80 &#176;C for 120 min using a commercially available curing agent (see Table of Materials). The curing agent enhances the mechanical properties of the printed MC device by exposing it to temperature and UV.</li>
	<li>Cure the MC devices thermally in an oven at 160 &#176;C for 180 min.</li>
	<li>Sterilize the devices using an autoclave for 45 min on a gravity cycle.</li>
</ol>
2. Hydrogel preparation
<ol>
	<li>Synthesize methacrylated hyaluronic acid (MAHA, 85%-115% methacrylation) and characterize using a protocol described by Seidlits et al.20. 
	NOTE: To support the growth of DRGs, triple hydrogels composed of methacrylated hyaluronic acid, type I collagen, and laminin are commonly used. Findings indicate that DRGs commonly grow in MAHA, ranging from 1.25-2.5 mg/mL and collagen from 2.0-4.5 mg/mL. For laminin, it is found that 0.75 mg/mL is enough to ensure robust growth of DRGs. Herein, methods to create a triple gel with 1.25 mg/mL MAHA, 4.5 mg/mL collagen, and 0.75 mg/mL laminin are detailed. Hydrogel preparation should be done in a laminar flow cabinet to provide an aseptic workspace.</li>
</ol>
3. Photoinitiator solution preparation
NOTE: A photoinitiator is necessary to crosslink the MAHA under UV light. It is commonly used at percentages from 0.3% to 0.6%.
<ol>
	<li>Prepare the photoinitiator solution 3 days prior to the DRG harvest.</li>
	<li>Pre-heat the sonicating water bath to 37 &#176;C before preparing the photoinitiator solution.</li>
	<li>Working with the sterile technique in a flow hood, prepare 23.125 mg/mL sodium bicarbonate (NaHCO3) solution by dissolving NaHCO3 in 5x Dulbecco&#39;s Modified Eagle Medium (DMEM)-HEPES solution.</li>
	<li>In a glass vial, mix 0.6% weight/volume (w/v) photoinitiator in a 20% vol/vol 1x sterile phosphate-buffered saline (PBS) and 80% vol/vol 5X DMEM/HEPES solution.</li>
	<li>To protect from light, wrap the glass vial in aluminum foil. However, ensure to remove the aluminum foil before transferring the vial to the sonicating water bath heated to 37 &#176;C with sonication between 30-40 min. 
	NOTE: Photoinitiator solution must be protected from light because it can break down into radicals and start the polymerization process when exposed to light21.</li>
	<li>Sterile filter the solution with a 0.22 &#181;m syringe filter into a new sterile glass vial.</li>
</ol>
4. Methacrylated hyaluronic acid dissolution
<ol>
	<li>Dissolve the methacrylated hyaluronic acid (prepared in step 2) on the same day the photoinitiator solution is prepared (step 3).</li>
	<li>Place the frozen lyophilized MAHA in a desiccator for 15 min to come to room temperature.</li>
	<li>Working in a laminar flow hood and using the sterile weighing technique, weigh the lyophilized MAHA in a glass vial.</li>
	<li>Add the necessary volume of filtered photoinitiator solution to MAHA to come to the desired MAHA concentration. The common final gel concentration is 1.25 mg/mL MAHA and 4.5 mg/mL collagen.</li>
	<li>Seal the cap with laboratory sealing film, wrap the glass vial with aluminum foil, and place it on an orbital shaker plate at room temperature until fully dissolved (usually 3 days). Occasionally, vortex the solution to break apart large clumps of MAHA to ensure uniform dissolution of MAHA.</li>
</ol>
5. Device assembly
<ol>
	<li>Place the MC device into a 48-well plate a day before the DRG harvest.</li>
	<li>Apply a thin line of silicone gel in the groove beneath the MC device (Figure 2B) using a 3 mL syringe. This will help the device to securely attach to the well plate.</li>
	<li>With the help of #3 forceps, gently place the MC device into the well of a 48-well plate, as shown in Figure 2C. 
	NOTE: The size of the device can be modified to suit the intended application, and a plate of a different size can be used if desired. The devices can be reused. To reuse, the hydrogel must be gently washed out from all tunnels, and the loading groove beneath the device must be used with 70% ethanol with gentle agitation. After this is complete, devices can be re-autoclaved to sterilize prior to use.</li>
</ol>
6. Animal preparation
<ol>
	<li>Autoclave all the necessary dissection tools prior to the start of harvest.</li>
	<li>Obtain an adult male or female Sprague Dawley rat aged 12-20 weeks. The sex of the rat does not impact DRG growth.</li>
	<li>Euthanize the rat according to the approved IACUC protocol using CO2 overdose as the primary method of euthanasia and bilateral pneumothorax puncture as a secondary method of euthanasia according to American Veterinary Medical Association Guidelines22.</li>
	<li>Place the rat on a sterile underpad on a dissecting table with the ventral side down. Spray the fur down with 70% ethanol.</li>
</ol>
7. Dorsal root ganglia (DRG) harvest
<ol>
	<li>Prepare trimming media containing 86% Neurobasal A media, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PS), 1% Glutamine, and 2% B27 plus 50x and transfer into a 24-well plate. This media and well-plate will be used for temporary storage of harvested DRGs prior to embedding them in the hydrogel.</li>
	<li>Wear a lab coat, surgical mask, bonnet, safety glasses, and gloves.</li>
	<li>Using large blunt-nose scissors, cut through the skin at the base of the cervical spine and then down the length of the spine.</li>
	<li>To further drain blood away from the spinal canal, cut underneath the shoulder blades and down through the muscle to sever major blood vessels.</li>
	<li>Use large, sharp-nose scissors to cut the muscle away from the spine. Clear as much muscle as possible to visualize the spine.</li>
	<li>Remove the dorsal and lateral segments of the vertebrae. Use a straight Rongeur and sharp-nosed scissors to remove the spinous and transverse processes of the vertebrae, starting at the cervical end and moving toward the lumbar end.</li>
	<li>Remove the spinal cord. Use small sharp-nose scissors to carefully cut through the nerve roots connected to the DRGs along both sides of the spinal cord. Cut the cervical end of the spinal cord and carefully lift out of the spinal canal gradually, cutting through any remaining nerve roots connected to the DRGs. 
	NOTE: Fine motor skills are essential to ensure the DRG body is not severed in the process.</li>
	<li>To expose DRGs, use the curved Rongeur to remove more of the lateral parts of the vertebrae. Pull outward from the midline, alternating sides. Be careful not to cut through the DRG body since it may cause damage to the cell bodies and axons.</li>
	<li>Using the #3 forceps and straight-edge spring scissors, remove DRGs in between each level of the vertebrae. Grip the spinal nerve roots from the DRG, carefully pull them out of the pocket, and cut through the other end of the spinal nerve. Be careful not to grip the DRG body directly.</li>
	<li>Place the DRGs in wells of a 24-well plate (containing trimming media, step 7.1) on ice.</li>
	<li>After collecting all DRGs, clean up the workspace, place the carcass in an autoclave bag, and store/dispose of it appropriately according to IACUC protocol.</li>
</ol>
8. DRG trimming and cutting
<ol>
	<li>Fill a 60 mm Petri dish on a clean bench with trimming media (step 7.1). Set up the dissecting scope and glass bead sterilizer and place an autoclaved toolbox with trimming tools (#3 forceps and straight-edge spring scissors) by the scope.</li>
	<li>Under the dissecting scope, remove excess tissue around the DRG and trim the nerve roots close to the DRG body (typically slightly darker than the surrounding nerves).</li>
	<li>Fill a second 60 mm Petri dish with fresh media. Under the dissecting scope, cut the trimmed DRGs to about 0.5 mm (or between 0.5 mm and 0.8 mm). A ruler is placed under the Petri dish during cutting to have the correct estimated size of cut DRGs.</li>
	<li>Transfer cut DRGs into a fresh 24-well plate with the media on ice. 
	NOTE: DRG trimming, cutting, and embedding should be done in a laminar flow cabinet to provide an aseptic workspace. In the absence of a laminar flow cabinet, aseptic techniques must be observed, with minimum appropriate personal protective equipment (PPE) recommended (lab coat, masks, goggles, and gloves) and all tools sterilized throughout the process to mitigate potential contamination.</li>
</ol>
9. Collagen neutralization
<ol>
	<li>Neutralize the collagen on the same day as the DRG harvest after trimming and cutting the DRGs.</li>
	<li>Working on ice, pipette ~8.16 % ultra-pure water, ~1.84% 1 M sodium hydroxide (NaOH), ~10% 10x PBS, and ~80% of collagen of the total volume of solution into a glass vial.</li>
	<li>Mix slowly using a sterile low-retention pipette tip to pipette collagen (usually added last) into the mixture. Ensure to keep the pipette tip in the solution while mixing to avoid creating bubbles. Check the pH of the neutralized collagen using pH strips to be sure it is ~7. 
	NOTE: Neutralized collagen can be kept on ice for 2-3 h without gelling; therefore, hydrogel fabrication should be carried out expeditiously once collagen is neutralized.</li>
</ol>
10. Hydrogel fabrication
<ol>
	<li>Working on ice, pipette laminin (to a final concentration of 0.75 mg/mL) and 1x PBS into a glass vial.</li>
	<li>Using sterile low retention pipette tips, pipette neutralized collagen and MAHA solution into the mixture to arrive at the desired concentration.</li>
	<li>Mix slowly and check the pH of the resulting hydrogel (should be ~7).</li>
</ol>
11. DRG embedding
<ol>
	<li>Perform multi-compartment embedding.
	<ol>
		<li>Pipette 65.1 &#181;L and 52.8 &#181;L of the pre-mixed hydrogel into the soma and neurite compartments, respectively. 
		NOTE: This assumes the use of MC devices in a 48-well plate. These volumes can be changed based on the size of the MC device used.</li>
		<li>Gently embed the cut DRGs into the soma compartment (Figure 2A), ensuring they do not scratch the bottom of the well plate. Place the DRG in the middle so the neurites can grow into the adjacent compartments through the tunnels.</li>
		<li>Thermal crosslink the hydrogel in the incubator at 37 &#176;C for 30 min. While this occurs, turn on the UV lamp to warm up.</li>
		<li>After thermal crosslinking, UV crosslink the hydrogel for 90 s.</li>
		<li>Add DRG growth media to the hydrogel and DRG and perform a full media change after an hour to eliminate unreacted or remaining traces of photoinitiator that persist in the media.</li>
		<li>Perform a half media change every 3 days and monitor the growth of the DRGs by viewing under the microscope.</li>
		<li>After ~4 weeks, when DRG neurites begin to grow (Figure 3A), capture images with the fluorescent plate imager and quantify the length of neurites using FIJI (ImageJ).</li>
	</ol>
	</li>
</ol>
12. Control DRG embedding
<ol>
	<li>Pipette 250 &#181;L of hydrogel in a 48-well plate (without MC).</li>
	<li>Gently place the cut DRG in the middle of the well and halfway down into the hydrogel, ensuring it does not scratch the bottom of the well plate.</li>
	<li>Thermal crosslink the hydrogel in the incubator at 37 &#176;C for 30 min.</li>
	<li>UV crosslink the hydrogel for 90 s.</li>
	<li>Add DRG growth media to the hydrogel and DRG and perform a full media change after an hour.</li>
	<li>Perform half media change every 3 days and monitor the growth of the DRG by viewing it under the microscope.</li>
	<li>After ~4 weeks, when DRG neurites begin to grow (Figure 3B), capture images with the fluorescent plate imager. 
	NOTE: Any brightfield microscope will work for imaging. An automated plate-based microscope makes this process faster. Control embedding in plain gels without the device is done to compare the growth of DRG neurites in multi-compartment. Once users validate that DRGs grow similarly in MC and control gels, they may not need to repeat control gels each time.</li>
</ol>
13. DRG imaging
<ol>
	<li>Capture brightfield images at 4x magnification using a fluorescent plate imager at a range of focal distances to visualize the entire DRG and MC Device.</li>
	<li>Adjust the brightness and contrast of the image to make it easier to view the neurites.</li>
	<li>Save the image as a .tiff file.</li>
</ol>
14. DRG neurite quantification
<ol>
	<li>Download and install FIJI (ImageJ).</li>
	<li>Open ImageJ. Open the image file and ensure that the file is monochrome.</li>
	<li>Enhance the contrast so that the smallest neurites are visible. Do this by opening the brightness and contrast option using the shortcut Ctrl+Shift+C.</li>
	<li>Adjust the sliders of the controller as needed to visualize the neurites, and click on Apply for the desired brightness and contrast.</li>
	<li>Open the neurite tracer by opening the Plugins menu, selecting Segmentation, and clicking on Simple Neurite Tracer.</li>
	<li>Start a new trace by clicking on one end of the neurite right up against the DRG body.</li>
	<li>Click another point along the neurite to add a trace until the neurite is traced end to end. Click on Finish Path after tracing the neurite.</li>
	<li>Convert the traced length to real units and save the results by copying and pasting them into Excel.</li>
	<li>Use the straight-line tool in FIJI to draw a line of the same length as the scale bar (not letting go of the end of the line) and look at the length value visible under the toolbar. The length value is used for the conversion.</li>
	<li>Measure the length of six long neurites extending from both sides of the DRG (Figure 3C,D) and calculate the average length of these neurites to give a representative average length of the DRG, as shown in Figure 4. 
	NOTE: Immunostaining has been tested on DRG explants in plain gels to show the presence of non-neuronal support cells7. DRGs are fixed in 4% paraformaldehyde (PFA) at room temperature, washed three times with 1x PBS for 15 min, and stored in 1x PBS at 4 &#176;C until immunostaining.</li>
	<li>For explants in MC devices, remove the MC device and follow the same process described above7.</li>
</ol>

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

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<li>Bian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+hyaluronic+acid+hydrogel+crosslinking+density+and+macromolecular+diffusivity+on+human+MSC+chondrogenesis+and+hypertrophy%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy.</a> Biomaterials. 34 (2), 413-421 (2013).</li>
<li>Haberberger, R. V., Barry, C., Dominguez, N., Matusica, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+dorsal+root+ganglia%5BTitle%5D)+AND+%22Front+Cell+Neurosci%22%5BJournal%5D)">Human dorsal root ganglia.</a> Front Cell Neurosci. 13, 271(2019).</li>
<li>Schwaid, A. G., Krasowka-Zoladek, A., Chi, A., Cornella-Taracido, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+the+rat+and+human+dorsal+root+ganglion+proteome%5BTitle%5D)+AND+%22Scientific+Rep%22%5BJournal%5D)">Comparison of the rat and human dorsal root ganglion proteome.</a> Scientific Rep. 8 (1), 13469(2018).</li>
</ol>

The authors of this study declare that they have no conflict of interest.

This protocol outlines a method to harvest adult Sprague Dawley DRGs and culture them in 3D natural hydrogels. In contrast to this method, other approaches to harvesting DRGs from mice and rats involve isolating the spinal column. The excised spinal column is halved, and the spinal cord is removed to expose DRGs23,24,25. Damage to the spinal cord limits blood supply, which affects DRGs and internal neurons26</s...

Chronic pain is the leading cause of disability and loss of work globally1. Chronic pain affects about 20% of adults globally and imposes a significant societal and economic burden2, with total costs estimated between $560 and $635 billion every year in the United States3.
The main peripheral feature exhibited by chronic pain patients is a lowered stimulation threshold of nerves, which leads to the nervous system being more responsive to stimuli4,5. The lowered stimulation threshold can result in a painful response to a previously non-painful stimulus (allodynia) or a heightened response to a painful stimulus (hyperalgesia)6. Current chronic pain treatments have limited efficacy, and treatments that succeed in animal models often fail in human trials due to mechanistic differences in pain manifestation7. In vitro models that can more accurately mimic peripheral sensitization mechanisms have the potential to increase the translation of new therapeutics8,9. Further, by modeling key aspects of sensitized nerves in a culture system, researchers could develop a deeper understanding of the mechanisms that drive lowered thresholds and identify novel therapeutic targets that reverse them10.
The ideal in vitro platforms or microphysiological systems would incorporate the physical separation of distal neurites and dorsal root ganglia (DRG) cell body, a three-dimensional (3D) cellular environment, and the presence of native support cells to closely mimic in vivo conditions. However, a recent paper by Caparaso et al.11 shows that current DRG culture platforms lack one or more of these key features, making them insufficient in replicating in vivo conditions. Even though these platforms are easy to set up, they do not mimic the biological basis of peripheral sensitization and thus may not translate to in vivo efficacy. To address this limitation, a physiologically relevant in vitro model has been developed for the culture of dorsal root ganglia (DRG) within a hydrogel matrix with three isolated compartments to allow temporal fluidic isolation of neurites and DRG cell bodies11. This model offers both physiological and anatomical relevance, which has the potential to study peripheral sensitization of neurons in vitro.
The growing interest in the use of DRG explants in a 3D culture is due to their ability to facilitate robust neurite growth, which serves as an indirect indicator of DRG viability12. While primary neonatal or embryonic DRG explants are predominantly used in current in vitro culture platforms13,14, using explants from adult rodents provides a better model of mature neuronal physiology, which closely mimics human DRG physiology compared to explants from neonatal or embryonic rodents15. Explant DRGs refer to the preservation of the cellular and molecular tissue of native DRG tissue, primarily by maintaining native non-neuronal support cells.Herein, this protocol describes the methodology to harvest and culture DRG explants from adult Sprague Dawley rats in a multi-compartment (MC) device (Figure 1).
Efficacy has been shown in culturing DRGs from the cervical, thoracic, and lumbar spine with no observable differences in neurite growth. For this application, the objective was to elicit neurite growth into the outer compartments of the device; therefore, this article did not discriminate among DRG levels. However, if needed for a specific experiment, the DRG level can be tailored to meet experimenters&#39; needs. There are currently other compartmentalized culture models for the 3D culture of DRGs16, however, these devices do not contain preserved native non-neuronal support cells, which can limit translation. Preserving the native structure of harvested DRGs is important because it ensures the retention of non-neuronal support cells, whose interactions with DRG neurons are essential for maintaining the functional properties of these neurons. Several studies co-cultured dissociated DRGs with non-native neuronal cells such as Schwann cells to promote myelination of neurons17,18,19.

<table><tbody><tr><td>#5 forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>For trimming and cutting DRG</td></tr><tr><td>10x DMEM</td><td>MilliporeSigma</td><td>D2429</td><td></td></tr><tr><td>1x PBS (autoclaved)</td><td>Prepared in lab</td><td></td><td>7.3 - 7.5 pH</td></tr><tr><td>24 well plates</td><td>VWR</td><td>82050-892</td><td>To temporarily store harvested and cut DRGs</td></tr><tr><td>3 mL Syringe sterile, single use</td><td>BD</td><td>309657</td><td></td></tr><tr><td>48 well plates</td><td>Greiner Bio-One</td><td>677180</td><td></td></tr><tr><td>60 mm Petri dish</td><td>Fisher Scientific</td><td>FB0875713A</td><td>To hold media for trimming and cutting</td></tr><tr><td>Aluminium foil</td><td>Fisherbrand</td><td>01-213-104</td><td></td></tr><tr><td>B27 Plus 50x</td><td>ThermoFisher</td><td>17504044</td><td>For DRG media</td></tr><tr><td>Collagen type I</td><td>Ibidi</td><td>50205</td><td></td></tr><tr><td>Curved cup Friedman Pearson Rongeur</td><td>Fine Science Tools</td><td>16221-14</td><td>For dissection</td></tr><tr><td>Dumont #3 forceps</td><td>Fine Science Tools</td><td>11293-00</td><td>For dissection</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher</td><td>16000044</td><td>For DRG media</td></tr><tr><td>Form cure</td><td>Form Labs</td><td></td><td>curing agent</td></tr><tr><td>Form wash</td><td>Form Labs</td><td></td><td>To wash excess resins off MC</td></tr><tr><td>Glass bead sterilizer</td><td>Fisher Scientific</td><td>NC9531961</td><td></td></tr><tr><td>Glass vials (8 mL)</td><td>DWK Life Sciences (Wheaton)</td><td>224724</td><td></td></tr><tr><td>GlutaMax</td><td>ThermoFisher</td><td>35050-061</td><td>For DRG media</td></tr><tr><td>HEPES (1M)</td><td>Millipore Sigma</td><td>H0887</td><td></td></tr><tr><td>High temp V2 resin</td><td>FormLabs</td><td>FLHTAM02</td><td></td></tr><tr><td>Hyaluronic Acid Sodium Salt</td><td>MilliporeSigma</td><td>53747</td><td>Used to make MAHA</td></tr><tr><td>Irgacure</td><td>MilliporeSigma</td><td>410896</td><td></td></tr><tr><td>Laminin</td><td>R&amp;amp;D Systems</td><td>344600501</td><td></td></tr><tr><td>Large blunt-nose scissors</td><td>Militex</td><td>EG5-26</td><td>For dissection</td></tr><tr><td>Large forceps (serrated tips)</td><td>Militex</td><td>9538797</td><td>For dissection</td></tr><tr><td>Large sharp-nosed scissors</td><td>Fine Science Tools</td><td>14010-15</td><td>For dissection</td></tr><tr><td>Low Retention pipette tips</td><td>Fisher Scientific</td><td>02-707-017</td><td>For pipetting collagen and MAHA</td></tr><tr><td>Methacrylated hyaluronic acid (MAHA)</td><td>Prepared in lab</td><td>N/A</td><td>85 - 115 % methacrylation</td></tr><tr><td>Nerve Growth Factor (NGF)</td><td>R&amp;amp;D Systems</td><td>556-NG-100</td><td>For DRG media</td></tr><tr><td>Neurobasal A Media</td><td>ThermoFisher</td><td>10888022</td><td>For DRG media</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>Penicillin/Streptomycin (PS)</td><td>EMD Millipore</td><td>516106</td><td>For DRG media</td></tr><tr><td>pH test strips</td><td>VWR International</td><td>BDH35309.606</td><td></td></tr><tr><td>Pipette tips (1000 &amp;micro;L)</td><td>USA Scientific</td><td>1111-2021</td><td></td></tr><tr><td>Preform 3.23.1 software</td><td>Formslab</td><td></td><td>To upload STL file</td></tr><tr><td>Rat</td><td>Charles River</td><td></td><td></td></tr><tr><td>Resin 3D printer</td><td>Form Labs</td><td>Form 3L</td><td>3D printing MC device</td></tr><tr><td>Small sharp-nosed scissors</td><td>Fine Science Tools</td><td>14094-11</td><td>For dissection</td></tr><tr><td>Sodium bicarbonate</td><td>MilliporeSigma</td><td>S6014</td><td></td></tr><tr><td>Straight cup rongeur</td><td>Fine Science Tools</td><td>16004-16</td><td>For dissection</td></tr><tr><td>Straight edge spring scissors</td><td>Fine Science Tools</td><td>15024-10</td><td>For dissection</td></tr><tr><td>Surgical Scaplel blade (No. 10)</td><td>Fisher Scientific</td><td>22-079-690</td><td></td></tr><tr><td>Syring filters, PES (0.22 &amp;micro;m)</td><td>Celltreat</td><td>229747</td><td></td></tr><tr><td>Tiny spring scissors</td><td>World Precision Instruments</td><td>14003</td><td>For trimming and cutting DRG</td></tr><tr><td>UV lamp</td><td>Analytik Jena US</td><td></td><td>To photocrosslink hydrogel (15 - 18 mW/cm2)</td></tr></tbody></table>

harvesting, embedding, and culturing dorsal root ganglia in multi-compartment devices to study peripheral neuronal features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

The present protocol described a technique to harvest and culture DRG from adult Sprague Dawley rats in a multi-compartment (MC) device. As shown&#160;in Figure 1, DRG harvested from adult rats was trimmed and cut into ~0.5 mm. The trimmed and cut DRGs were then embedded in a hydrogel in the soma region of the MC device (Figure 2) and cultured for 27 days before neurite quantification. DRG was cultured in plain gel to serve as the control. The c...

Author Spotlight: In Vitro and In Vivo Models to Enhance Chronic Pain Treatment Efficacy

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

harvesting-embedding-culturing-dorsal-root-ganglia-multi-compartment

Research

JoVE Journal

Bioengineering

928 Views.  University of Nebraska-Lincoln. This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

Video: Author Spotlight: In Vitro and In Vivo Models to Enhance Chronic Pain Treatment Efficacy

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

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.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Cancer Research

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 ...

Biology

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

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

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.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Neuroscience

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

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

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

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.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

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 (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Harvesting DRG from Rat Spine: A Procedure to Extract Dorsal Root Ganglion from the Spine of a Rat Model

Source: Schmidt, L. B. et al. <a href="https://www.jove.com/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the extraction of dorsal root ganglion or DRG from a harvested rat spine for downstream applications.

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

Assays & techniques

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

Source: de Luca, A. C., et al., <a href="https://app.jove.com/v/52543">Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration.</a> J. Vis. Exp. (2015).
This video demonstrates a method for harvesting neurons from the dorsal root ganglia (DRG). DRG from a rat&#39;s spinal column is isolated, treated with enzymes, and then mechanically dissociated to obtain a single-cell suspension. The neurons are separated from glial cells using density gradient centrifugation and cultured in a growth medium.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Set-up

	Prior to ...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

To harvest the dorsal root ganglia, transfer the first embryo into a 10-centimeter culture dish containing room-temperature PBS under a dissecting microscope in the prone position and insert micro-dissecting forceps along either side of the spinal cord.
Using blunt dissection, separate the spinal cord from the surrounding soft tissue, using forceps to excise the spinal cord along the neck opening and tail stub. Remove the residual soft tissue from the freed spinal cord until only the spinal cord, nerve roots, and attached dorsal root ganglion remain.
Detach the individual dorsal root ganglia from their connecting nerve roots. Then, use a pipette pen equipped with a 1-milliliter pipette tip to transfer up to 100 dorsal root ganglia into a 1.5-milliliter microcentrifuge tube of PBS.
Collect the ganglia by centrifugation, and re-suspend the pellets in 200 microliters of recombinant enzymatic cell dissociation reagent per tube. After 10 minutes at 37 degrees Celsius, centrifuge the dorsal root ganglia again using a 200-microliter pipette tip to gently triturate the pellets in dorsal root postganglionic neuron maintenance medium.
Seed the dorsal root ganglion cells at 5 x 103 cells per square centimeter onto Poly-D-Lysine laminin-coated six-well plates in 1.5 milliliters of dorsal root ganglion neuron maintenance medium per well.
After two days in culture at 37 degrees Celsius and 5% CO2, wash the cells and return the neuronal cell cultures to the incubator in fresh dorsal root ganglion neuron purification medium for another two days.
After 3 to 4 maintenance and purification cycles, the dorsal root ganglion cell cultures should test positive for the neuronal marker Tuj-1 and negative for the glial cell marker S100 beta.
On day 7 of the Schwann cell-like cell culture, rinse the cells in PBS, followed by dissociation with 0.5 milliliters of recombinant enzymatic cell dissociation reagent per well at 37 degrees Celsius for 5 minutes. Resuspend the cell pellet in co-culture medium and seed the Schwann cell-like cells onto the purified dorsal root ganglion neuron culture at 1 x 103 cells per square centimeter for 14 days of co-culture at 37 degrees Celsius and 5% CO2.

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

Summary

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Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

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

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

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Harvesting DRG from Rat Spine: A Procedure to Extract Dorsal Root Ganglion from the Spine of a Rat Model

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