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 
...

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

<ol>
	<li>Vos, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+regional,+and+national+incidence,+prevalence,+and+years+lived+with+disability+for+310+diseases+and+injuries,+1990-2015:+A+systematic+analysis+for+the+global+burden+of+disease+study+2015.">Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990-2015: A systematic analysis for the global burden of disease study 2015.</a> Lancet. 388 (10053), 1545-1602 (2016).</li><li>Goldberg, D. S., Mcgee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain+as+a+global+public+health+priority.">Pain as a global public health priority.</a> BMC Public Health. 11 (1), 770 (2011).</li><li>Gaskin, D. J., Richard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+economic+costs+of+pain+in+the+United+States.">The economic costs of pain in the United States.</a> J Pain. 13 (8), 715-724 (2012).</li><li>Kim, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupled+activation+of+primary+sensory+neurons+contributes+to+chronic+pain.">Coupled activation of primary sensory neurons contributes to chronic pain.</a> Neuron. 91 (5), 1085-1096 (2016).</li><li>Im, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+sensory+neurons+and+spinal+response+to+an+experimental+osteoarthritis+pain+model.">Alteration of sensory neurons and spinal response to an experimental osteoarthritis pain model.</a> Arthritis Rheum. 62 (10), 2995-3005 (2010).</li><li>Jensen, T. S., Finnerup, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allodynia+and+hyperalgesia+in+neuropathic+pain:+Clinical+manifestations+and+mechanisms.">Allodynia and hyperalgesia in neuropathic pain: Clinical manifestations and mechanisms.</a> Lancet Neurol. 13 (9), 924-935 (2014).</li><li>Berge, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+validity+of+behavioral+animal+models+for+chronic+pain.">Predictive validity of behavioral animal models for chronic pain.</a> Br J Pharmacol. 164 (4), 1195-1206 (2011).</li><li>Anderson, W. A., Willenberg, A. R., Bosak, A. J., Willenberg, B. J., Lambert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+capillary+alginate+gel+(capgel&#8482;)+to+study+the+three-dimensional+development+of+sensory+nerves+reveals+the+formation+of+a+rudimentary+perineurium.">Use of a capillary alginate gel (capgel&#8482;) to study the three-dimensional development of sensory nerves reveals the formation of a rudimentary perineurium.</a> J Neurosci Methods. 305, 46-53 (2018).</li><li>Mohammed Izham, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+possibilities+of+using+in+vitro+model+for+neuropathic+pain+studies.">Exploring the possibilities of using in vitro model for neuropathic pain studies.</a> Neurosci Res Notes. 5, 144 (2022).</li><li>Park, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+in+vitro+model+of+the+peripheral+nervous+system.">A three-dimensional in vitro model of the peripheral nervous system.</a> NPG Asia Mater. 13 (1), 2 (2021).</li><li>Caparaso, S. M., Redwine, A. L., Wachs, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+multi-compartment+in+vitro+model+for+dorsal+root+ganglia+phenotypic+assessment.">Engineering a multi-compartment in vitro model for dorsal root ganglia phenotypic assessment.</a> J Biomed Mater Res B Appl Biomater. 111 (11), 1903-1920 (2023).</li><li>Krug, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+human+neurite+growth+assay+as+specific+screen+for+developmental+neurotoxicants.">Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.</a> Arch Toxicol. 87 (12), 2215-2231 (2013).</li><li>F, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+stiffness+negatively+affects+axon+elongation,+growth+cone+area+and+F-actin+levels+in+a+collagen+type+I+3D+culture.">Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F-actin levels in a collagen type I 3D culture.</a> J Tissue Eng Regen Med. 16 (2), 151-162 (2021).</li><li>Spearman, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+methacrylated+hyaluronic+acid-based+hydrogels+as+scaffolds+for+soft+tissue+engineering+applications.">Tunable methacrylated hyaluronic acid-based hydrogels as scaffolds for soft tissue engineering applications.</a> J Biomed Mater Res A. 108 (2), 279-291 (2020).</li><li>Zhu, W., Oxford, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+expression+of+neonatal+and+adult+DRG+neurons+correlates+with+the+differential+sensitization+of+TRPV1+responses+to+nerve+growth+factor.">Differential gene expression of neonatal and adult DRG neurons correlates with the differential sensitization of TRPV1 responses to nerve growth factor.</a> Neurosci Lett. 500 (3), 192-196 (2011).</li><li>Van De Wijdeven, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structuring+a+multi-nodal+neural+network+in+vitro+within+a+novel+design+microfluidic+chip.">Structuring a multi-nodal neural network in vitro within a novel design microfluidic chip.</a> Biomed Microdevices. 20, 1-8 (2018).</li><li>Chan, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43 (2), 183-191 (2004).</li><li>Ng, B. K., Chen, L., Mandemakers, W., Cosgaya, J. M., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterograde+transport+and+secretion+of+brain-derived+neurotrophic+factor+along+sensory+axons+promote+Schwann+cell+myelination.">Anterograde transport and secretion of brain-derived neurotrophic factor along sensory axons promote Schwann cell myelination.</a> J Neurosci. 27 (28), 7597-7603 (2007).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+exerts+contrasting+effects+on+peripheral+myelination+of+NGF-dependent+and+BDNF-dependent+drg+neurons.">BDNF exerts contrasting effects on peripheral myelination of NGF-dependent and BDNF-dependent drg neurons.</a> J Neurosci. 29 (13), 4016-4022 (2009).</li><li>Seidlits, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+hyaluronic+acid+hydrogels+with+tunable+mechanical+properties+on+neural+progenitor+cell+differentiation.">The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation.</a> Biomaterials. 31 (14), 3930-3940 (2010).</li><li>Tomal, W., Ortyl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-soluble+photoinitiators+in+biomedical+applications.">Water-soluble photoinitiators in biomedical applications.</a> Polymers. 12 (5), 1073 (2020).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Avma guidelines for the euthanasia of animals: 2013 edition. , (2013).</li><li>Sleigh, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dissection+and+culture+of+murine+dorsal+root+ganglia+neurons+to+study+neuropeptide+release.">Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.</a> STAR Protocols. 2 (1), 100333 (2021).</li><li>Bai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+anatomical+rat+atlas.">A high-resolution anatomical rat atlas.</a> J Anatomy. 209 (5), 707-708 (2006).</li><li>Bian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+hyaluronic+acid+hydrogel+crosslinking+density+and+macromolecular+diffusivity+on+human+MSC+chondrogenesis+and+hypertrophy.">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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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&#38;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 &#181;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 &#181;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.

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

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

Our research seeks to understand some of the underlying mechanisms that contribute to chronic musculoskeletal pain, so we can develop novel targeted treatments. Thus, we have developed in vitro and in vivo models of chronic low back pain and neuronal sensitization. Current therapeutics that work in vivo in animal models often fail clinical trials due to mechanistic differences between animal models and human subject chronic pain phenotype.Individual test by that model, nociceptor hypersensitivity present in chronic pain have the potential to identify more promising treatments for chronic pain in humans. Our protocol clearly demonstrates how to isolate primary dorsal root ganglia, and culture them in a multi compartment device. By keeping intact primary dorsal root ganglia, we maintain native support cells and culture them in a multi compartment device, allowing anatomically relevant isolation between neuronal somas and peripheral neurites.This tool will help to culture primary dorsal root ganglia in a more physiologically and anatomically relevant way, thereby increasing translational efficacy of findings. Specifically, we are excited to use this system to identify novel treatment for nociceptor hypersensitivity present in chronic 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...

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.

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

Assembly of Multi-Compartment Device and Harvesting of Dorsal Root Ganglia (DRG) from Adult Sprague Dawley Rats

To begin, use a three milliliter syringe to apply a thin line of silicone gel into the groove beneath the MC device. Using number three forceps gently position the MC device into the well of a 48 well plate. Place the device into a 48 well plate one day before harvesting the dorsal root ganglion.Place the euthanized rat on the dissection platform. Using large blunt nose scissors, make an incision through the skin at the base of the cervical spine and extend it down the length of the spine. To further drain blood from the spinal canal, cut beneath the shoulder blades and through the muscle to sever major blood vessels.Using large sharp nose scissors, cut the muscle away from the spine, then remove the dorsal segments of the vertebrae. Use straight rongeur and sharp nose scissors to remove the spinous and transverse processes of the vertebrae starting at the cervical end and moving towards the lumbar end. Afterward, remove the spinal cord.Using small sharp nose scissors, carefully sever the nerve roots connected to the dorsal root ganglia along both sides of the spinal cord. Now cut the cervical end of the spinal cord and gradually lift it out of the spinal canal, severing any remaining nerve roots connected to the dorsal root ganglia. To expose the dorsal root ganglia, use a curved rongeur to remove additional lateral parts of the vertebrae.Using number three forceps and straight edge spring scissors, extract the dorsal root ganglia between each vertebra level. Grip the spinal nerve roots from the dorsal root ganglia. Gently pull them out of the pocket and cut through the other end of the spinal nerve.Lastly, place the dorsal root ganglia into wells of a 24 well plate on ice.

Trimming, Cutting, and Embedding the Dorsal Root Ganglion for Culturing in a Multi-Compartment (MC) Device

To begin, take the harvested dorsal root ganglia. On a clean bench, fill a 60-millimeter Petri dish with trimming media. Arrange the dissecting scope and glass bead sterilizer, and place an autoclave toolbox with trimming tools next to the scope.Using the dissecting scope, remove any excess tissue around the dorsal root ganglion, and trim the nerve roots close to the dorsal root ganglion body. Now, fill a second 60-millimeter Petri dish with fresh media. Using the dissecting scope, cut the trimmed dorsal root ganglia to approximately 0.5 millimeters.Transfer the cut dorsal root ganglia into a fresh 24-well plate containing media on ice. Then, pipette 65.1 and 52.8 microliters of the premixed hydrogel into the soma and neurite compartments, respectively. Carefully embed the cut dorsal root ganglia into the soma compartment.Thermally cross-link the hydrogel in the incubator at 37 degrees Celsius for 30 minutes. Then, UV cross-link the hydrogel for 90 seconds. Afterward, add dorsal root ganglia growth media to the hydrogel and dorsal root ganglia.Perform a complete media change after one hour to remove any unreactive or residual photo-initiator in the media.

Quantification of Dorsal Root Ganglion Neurite Growth in a Multi-Compartment (MC) Device Using Fiji

After imaging the dorsal root ganglion cultured in a multi-compartment device, launch Image J and open the image file. To enhance the contrast and make the smallest neurites visible using the shortcuts Control with Shift and C, open the Brightness and Contrast option. Adjust the brightness and contrast sliders to visualize the neurites.Then click on Apply. Now, navigate to Plugins, select Segmentation, and click Simple Neurite Tracer to open the neurite tracer. Then, click on one end of the neurite at the dorsal root ganglion body to initiate a new trace, and continue adding points along the neurite until it is fully traced.Then click on Complete Path. Using the straight line tool in Fiji, draw a line of the same length as the scale bar without releasing the end of the line. And note the length value under the toolbar.Set the scale to convert the neurite length to real units and copy the results to Excel. Then, convert the lengths to real units in Excel. Then, measure the length of six long neurites extending from both sides of the dorsal root ganglion, and calculate the average length and standard deviation of the dorsal root ganglion.On days 27 and 21, there was robust growth of neurites in the multi compartment and plain gels, respectively. The average length of neurites in the multi compartment device was comparable to neurite length in control plane gels.

314 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.

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

Summary