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. N., Weir, G. A., Schiavo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+step-by-step+dissection+protocol+for+the+rapid+isolation+of+mouse+dorsal+root+ganglia.">A simple, step-by-step dissection protocol for the rapid isolation of mouse dorsal root ganglia.</a> BMC Res Notes. 9 (1), 82 (2016).</li><li>Naveed, 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=Simple+methods+of+dissection+protocols+for+the+rapid+isolation+of+rat+dorsal+root+ganglia+under+the+non-microscopic+condition.">Simple methods of dissection protocols for the rapid isolation of rat dorsal root ganglia under the non-microscopic condition.</a> Neuroscience Research Notes. 6 (2), 212 (2023).</li><li>Perner, C., Sokol, C. 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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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