Name: Establishing In Vitro Models of Dorsal Root Ganglia Culture: Complementary Approaches for Investigating Cancer-Nerve Crosstalk (Video) | JoVE Journal
Uploaded: 2025-07-11T16:10:00.000+00:00
Duration: 11 min 26 s
Description: Learn to establish complementary 3D and 2D dorsal root ganglia cultures from a single mouse for studying cancer-nerve crosstalk. This protocol enables investigation of neuro-guided tumor migration and therapeutic strategies.

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5 minutes

This work was supported by operating grants from the Canadian Institutes for Health Research (PJT-178274) (LMM), and by an Ontario Graduate Scholarship (LCBO), and by a scholarship from the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Finance Code 001 (ERP). LMM is the Bracken Chair in Genetics and Molecular Medicine at Queen&#39;s University. Some images were generated in part using Biorender.

All mouse work was reviewed and approved by the Office of the University Animal Care Committee at Queen&#39;s University (Protocol number 2524). Animals were maintained and euthanized for tissue collection in compliance with Canadian Council on Animal Care, and National Centre for the Replacement, Refinement &#38; Reduction on Animals in Research (UK) ethical guidelines. In this protocol, we used tissue isolated from C57BL/6 animals but also successfully used other genotypes, including Rag2IL2R&#947;c KO animals. Sections 1-6 can be performed in a clean environment on a surface-sterilized lab bench (see Figure 1), while sections 7-9 must be performed in a biological safety cabinet. For better understanding, schematic representations of the steps mentioned were created (Figure 1).
1. Preparation of stock solutions
<ol>
	<li>Prepare Papain solution and Collagenase IV/Dispase II solution. Dissolve Papain in sterile PBS (Phosphate-buffered Saline) at a concentration of 30 U/mL; store in 1 mL aliquots at -20 &#176;C. Dissolve Collagenase Type IV at 1.25 mg/mL and Dispase II at 2.5 mg/mL in PBS. Store in 1 mL aliquots at -20 &#176;C.</li>
	<li>Prepare Neurobasal culture media (F12) supplemented with 2% B27 Supplement (50x stock solution), 10% fetal bovine serum (FBS), and 1% Penicillin-Streptomycin (Pen-Strep) (100x stock solution).</li>
	<li>Prepare Hank&#39;s Balanced Salt Solution (HBSS) with 1% Penicillin-Streptomycin (100x stock solution). 
	NOTE: To support initial cell survival and attachment, F12 media supplemented with 10% FBS is used to improve the establishment of the cultures. However, FBS also promotes the proliferation of non-neuronal cells, including fibroblasts. Reduced or serum-free media conditions (e.g., F12 supplemented with B27 only) may be used to minimize non-neuronal cell growth in the DRG culture and cancer co-culture but may affect survival, particularly of dissociated neuron cultures. Serum-free media should also be considered when collecting DRG conditioned media to assess its effects on cancer cell behavior.</li>
</ol>
2. Preparing reagents and materials for the dissection of DRGs (Figure 1A)
<ol>
	<li>Autoclave surgical instruments. Surface-sterilize the dissecting microscope with 70% ethanol and set up on a clean bench.</li>
	<li>Add 6-8 mL of HBSS-Pen-Strep (step 1.3) to two 10 cm culture plates and place on ice. 
	NOTE: These plates will be used to collect spinal columns after dissection. Depending on the number of spinal cords being dissected, additional plates might be needed.</li>
	<li>Add 8-10 mL of HBSS-Pen-Strep to a 15 mL conical tube, and place on ice.</li>
	<li>Add 3 mL of HBSS-Pen-Strep to a 3 cm culture plate, and place on ice. 
	NOTE: The 15 mL tube prepared in step 2.3 will be used to collect DRGs for dissociation, while the 3-cm culture plate (step 2.4) will be used to collect DRGs for the whole-mount model.</li>
</ol>
3. Preparing reagents and materials for whole-mount DRG culture
NOTE: Some steps in this section might require overnight preparation. Although any type of tissue culture plate can be used for DRG cultures, the volumes and instructions in this protocol were optimized for 8-well glass bottom slides, which provided optimal imaging quality for our analyses. Keep 8-well glass bottom slide, pipette tips, and Matrigel on ice at all times.
<ol>
	<li>Thaw Matrigel solution at 4 &#176;C overnight (or at least 3 h) before use. Make sure to keep Matrigel covered with ice at all times.</li>
	<li>Chill pipette tips and 8-well glass bottom slide at -20 &#176;C overnight to minimize Matrigel sticking to the tips and prevent premature Matrigel polymerization in the plate.</li>
	<li>Warm supplemented media (step 1.2) to 37 &#176;C in a water bath.</li>
</ol>
4. Preparing reagents and materials for dissociated DRG culture
<ol>
	<li>Thaw Papain solution and Collagenase IV/Dispase II solution (step 1.1) in a water bath at 37 &#176;C until ready for use. 
	NOTE: For each mouse dissected, use 1 aliquot (1 mL) each of Papain and Collagenase IV/Dispase II solution.</li>
	<li>Warm supplemented culture media (step 1.2).</li>
</ol>
5. Harvesting the spinal column (Figure 1B) and dissecting the DRGs (Figure 1C)
<ol>
	<li>Euthanize 4-8-week-old mice by isoflurane overdose followed by cervical dislocation. Lay the euthanized animal on its abdomen and shave its fur, focusing on the area over the spinal column (Figure 1B, steps 1,2).</li>
	<li>Spray the back of the animal with 70% ethanol and remove any loose fur, wiping it with paper towels from tail to skull to prevent fur from contaminating the dissection.</li>
	<li>Using sterilized scissors, make a straight incision along the shaved dorsal surface, from the tail to the skull. Retract the skin on both sides of the incision using blunt forceps to expose the spinal column (Figure 1B, step 3).</li>
	<li>Using a fresh pair of forceps and scissors to prevent contamination with fur, make a horizontal incision at the base (tail end) of the spinal column (Figure 1B, step 4).</li>
	<li>Make two parallel longitudinal incisions on each side of the spinal column to release it. Use forceps to gently lift the spinal column to remove the surrounding connective tissue (Figure 1B, step 5).</li>
	<li>To fully release the spinal column, make an incision at the top of the spinal column (proximal to the skull). Transfer the spinal column to the prechilled 10 cm plates containing HBSS-Pen-Strep on ice (step 2.2) (Figure 1C, step 6). 
	NOTE: It is convenient to collect all of the spinal columns in the same 10 cm plate and move individual columns one at a time to a separate plate for further dissection steps.</li>
</ol>
6. Dissecting the Dorsal Root Ganglia (DRGs)
<ol>
	<li>Using spring scissors, remove any residual connective tissue around the spinal column, exposing the vertebrae (Figure 1C, step 7). This allows clearer visualization of the vertebra and facilitates cutting along the midline. Collect trimmed columns in a fresh, prechilled 10 cm plate (step 2.2).</li>
	<li>One at a time, transfer the trimmed spinal cord to the microscope stage for dissection. Carefully split the spinal column in the sagittal plane using spring scissors (Figure 1D, step 8).</li>
	<li>Using fine-tipped forceps, remove the spinal cord from the spinal column, exposing the DRGs bilaterally along the roots of the spinal nerves (Figure 1D, step 9). 
	NOTE: Extra caution should be taken in this step as DRGs can be removed with the spinal cord if it is handled too roughly.</li>
	<li>Cut the spinal nerves with spring scissors and remove any connective tissue around the DRGs (Figure 1D, step 10). 
	NOTE: Removing connective tissue in this step minimizes debris in the DRG cultures.</li>
	<li>Using fine-tipped forceps, gently pull the DRG to extract it from the dorsal root. For whole mount culture, place the DRGs in the 3 cm plate (prepared in step 2.4) (Figure 1D, step 11). Pool the remaining DRGs in a 15 mL conical tube (prepared in step 2.3) to be used for dissociation. 
	NOTE: Keep plates and tubes on ice throughout the dissection process.</li>
	<li>Repeat steps 6.4 to 6.5 until all the DRGs are extracted. Repeat all the steps above for any additional spinal columns. 
	NOTE: Mice have 30-31 pairs of DRG depending on the strain, which will allow for the collection of up to 60 DRGs from each animal. If collecting DRGs only from the lumbar portion of the spinal cord, 10 DRGs can be extracted. Using DRGs from the same animal to establish both culture models reduces intra-experimental variability and ensures consistent and comparable results between complementary experiments. 
	NOTE: All the steps following the extraction of the DRGs must be performed in a biological safety cabinet.</li>
</ol>
7. Whole-mount DRG culture (Figure 2)
<ol>
	<li>Using prechilled tips (step 3.2), pipette 2 &#181;L of chilled Matrigel to form a droplet in each well of an 8-well glass bottom slide placed on ice (Figure 2A, step 1). To prevent Matrigel from polymerizing prematurely before adding the DRG, pipette only a single droplet at a time. To prevent scratching of the slide surface, place the slide in a 10 cm plate before placing on ice.</li>
	<li>Using fine-tipped forceps, transfer one DRG to a dry plate and gently move it around to remove any excess HBSS, preventing further dilution of the Matrigel when adding the DRG to the Matrigel droplet. Transfer the DRG to the Matrigel drop (Figure 2A, step 2). 
	NOTE: Use fine-tipped forceps to gently push the DRG into the Matrigel droplet, ensuring that the DRG is fully immersed and centrally positioned within the droplet.</li>
	<li>Repeat steps 7.1 to 7.2 for each DRG added to a Matrigel droplet.</li>
	<li>Incubate the 8-well glass bottom slide at 37 &#176;C for 5 min in a humidified incubator to allow the Matrigel to polymerize (Figure 2A, step 3).</li>
	<li>Carefully add 200 &#181;L of warm supplemented culture media to each well and incubate at 37 &#176;C with 5% CO2 (Figure 2B, step 4). Replace the media with fresh supplemented culture media every 72 h.</li>
	<li>Monitor the DRG using brightfield microscopy to assess its viability and neuronal outgrowth every 24 h (Figure 2C and Figure 3). 
	NOTE: Viability can be indicated by an increase in length and density of neurites. Images can be captured over time to demonstrate the development of the DRG cell extensions (Figure 3).</li>
</ol>
8. Dissociated DRG culture (Figure 4)
NOTE: Any multiwell plate can be used to seed the dissociated DRG neurons. However, in this protocol, 8-well glass-bottom slides were used because they provided better cell confluency and imaging quality for immunofluorescence analyses.
<ol>
	<li>Coating culture plates with collagen
	<ol>
		<li>Prepare a 50 &#181;g/mL working solution of acidified collagen I in sterile 0.01 M HCl in a biological safety cabinet.</li>
		<li>Pipette 100 &#181;L of collagen I solution into each well of the 8-well glass bottom slide. Tilt and gently rotate the slide to ensure the collagen solution covers the entire bottom surface of each well evenly. Incubate the slide at 37 &#176;C in a humidified incubator for 1 h to ensure collagen I polymerization. 
		NOTE: During incubation time, proceed with steps 8.2 and 8.3.</li>
		<li>After incubation, gently pipette off any residual unpolymerized collagen I solution from each well. Rinse each well with 100 &#181;L of PBS. Pipette off excess PBS, leaving a thin residual layer to prevent the collagen from drying out. 
		NOTE: The 8-well glass bottom slide can remain in the biological safety cabinet until the dissociated DRGs are ready for the seeding step.</li>
	</ol>
	</li>
	<li>Enzymatic digestion
	<ol>
		<li>Gently centrifuge the 15 mL tube containing extracted DRGs (step 6.5) for 5 min at 200 &#215; g at room temperature to pellet the DRGs. Gently remove the HBSS using a pipette.</li>
		<li>Rinse DRGs in prewarmed supplemented media, gently tapping the tube to ensure all the contents are being washed and centrifuge for 5 min at 200 &#215; g to pellet the DRGs.</li>
		<li>Pipette off the media and add 1 mL of Papain solution (Figure 4A, step 1). Mix gently and incubate for 20 min at 37 &#176;C in a tissue culture incubator. Gently shake the tube to agitate it every 5 min.</li>
		<li>After 20 min, add 4.5 mL of supplemented media and mix gently to neutralize Papain. Centrifuge tube for 5 min at 400 &#215; g to pellet DRGs. Carefully remove the supernatant using a pipette.</li>
		<li>Add 1 mL of Collagenase IV/Dispase II solution and incubate for 20 min at 37 &#176;C in the incubator. Gently shake the tube every 5 min (Figure 4A, step 1).</li>
		<li>After 20 min, add 4.5 mL of supplemented media and gently mix to neutralize the Collagenase IV/Dispase II solution. Centrifuge tube for 5 min at 400 &#215; g to pellet DRGs. Carefully remove the supernatant using a pipette.</li>
		<li>Add 2 mL of supplemented media and add DNase I to a final concentration of 0.2 mg/mL. At this point, the DRGs are thoroughly digested and may appear as a loose clump (Figure 4A, step 2).</li>
	</ol>
	</li>
	<li>Mechanical dissociation
	<ol>
		<li>Gently triturate the DRG, pipetting up and down 4-5x using a 1000 &#181;L filter pipette tip (Figure 4A, step 2).</li>
		<li>Perform a second trituration step using a 200 &#181;L pipette tip 4-5x. After this step, the sample appears cloudy with some visible clumps of tissue remaining (Figure 4A, step 2).</li>
		<li>Filter the cell suspension through a 70 &#181;m cell strainer into a 50 mL conical tube. Gradually rinse the strainer with 10 mL of media to ensure complete filtration of the cells through the strainer (Figure 4A, step 3).</li>
		<li>Centrifuge tube for 5 min at 1,000 &#215; g to pellet cells. Remove media from pellet and resuspend in 500 &#181;L of supplemented media (Figure 4A, step 4).</li>
		<li>Remove any residual PBS from wells of the 8-well glass bottom slide. Distribute the dissociated DRG suspension between the collagen-coated wells. Incubate the slides at 37 &#176;C in a humidified incubator with 5% CO2. Add more media as needed and replace media with fresh supplemented culture media every 48 h (Figure 4B, step 5). 
		NOTE: DRG-dissociated cells can be counted using a hemocytometer or cell counting chamber and trypan blue staining to ensure a consistent number of cells per well. For this protocol, approximately 1 &#215; 105 cells were seeded in each well.</li>
		<li>Monitor DRG-derived cells using brightfield microscopy to assess viability and neuronal outgrowth (Figure 4C and Figure 5). 
		NOTE: Neurons appear as rounded cell bodies with defined borders and may take 24 h or more to adhere, while non-neuronal cells typically settle before the neurons. Some neurite outgrowth might be observed after 24 h and increases over time up to 7 days in culture. Dissociated DRG-derived cells and whole-mount DRG are preincubated for 72 h before adding cancer cells and establishing co-cultures.</li>
	</ol>
	</li>
</ol>
9. Preparing cancer cell suspensions and establishing co-cultures
NOTE: Adherent cancer cells of many kinds can be used. Growth media and cell numbers for co-cultures will be cell line-dependent. We recommend preliminary testing before the experiment to determine timing for cell collection and optimal cell seeding numbers. The protocol described here was based on the use of human pancreatic cancer cell lines PanC1, and MiaPaCa2, and prostate cancer cell line DU-145.
<ol>
	<li>Wash cells with serum-free media and trypsinize them for approximately 3 min with 2 mL of trypsin/10 cm plate.</li>
	<li>Neutralize the trypsin by adding 6 mL of supplemented media and gently pipette up and down to ensure a single-cell suspension. Count the cells using a hemocytometer and trypan blue stain.</li>
	<li>Suspend the cancer cells at a concentration of 2 &#215; 105&#160;cells/mL in supplemented media (step 1.2). 
	NOTE: Optimal cell density may vary depending on cell size, doubling time, and the duration of co-culture and can be adjusted as needed.</li>
	<li>Whole-mount DRG and cancer cell co-culture (Figure 6B)
	<ol>
		<li>Aspirate the media off the whole mount DRG cultures in the 8-well glass bottom slide.</li>
		<li>Gently pipette 200 &#181;L of the cancer cell suspension (step 9.3) on top of the Matrigel droplet containing the whole mount DRG. 
		NOTE: This ensures a uniform distribution of cancer cells around the droplet. Be careful not to disturb the Matrigel droplet with the pipette tip.</li>
		<li>Incubate the co-culture slide in a humidified incubator at 37 &#176;C with 5% CO2. Refresh media daily by washing with 200 &#181;L of warmed supplemented media once, then adding 300 &#181;L of supplemented media to each well. 
		NOTE: Gently aspirate the media off using a pipette to avoid disturbing DRG-cancer cells co-culture. Over time, as the co-cultures become more complex and organized, they also become more susceptible to disturbances that may impact cellular interactions and co-culture integrity.</li>
		<li>Monitor the co-culture using brightfield or fluorescence microscope at 24 h intervals to assess cancer cell behavior and neuronal interactions (Figure 6B). 
		NOTE: Observation frequency can be adjusted based on the experimental timeline and cell interactions of interest.</li>
	</ol>
	</li>
	<li>Dissociated DRG and cancer cell co-culture (Figure 6C)
	<ol>
		<li>Aspirate the media off the dissociated cultures in the 8-well glass-bottom slide.</li>
		<li>Gently distribute 200 &#181;L of the cancer cell suspension (step 9.3) into each well. Follow the same incubation, media refreshment, and monitoring steps as outlined for whole-mount co-cultures (steps 9.4.3 and 9.4.4). 
		NOTE: For better visualization of cellular processes and cancer cell-neuron interactions, fluorescent cell trackers can be added to the culture, following the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>

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<li>Aisenbrey, E. A., Murphy, W. L., Aisenbrey, E. A., Murphy, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+alternatives+to+matrigel%5BTitle%5D)+AND+%22Nat+Rev+Mater%22%5BJournal%5D)">Synthetic alternatives to matrigel.</a> Nat Rev Mater. 5 (7), 539-551 (2020).</li>
<li>Berkovitch, Y., Seliktar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Semi-synthetic+hydrogel+composition+and+stiffness+regulate+neuronal+morphogenesis+-+pubmed%5BTitle%5D)+AND+%22Int+J+Pharm%22%5BJournal%5D)">Semi-synthetic hydrogel composition and stiffness regulate neuronal morphogenesis - pubmed.</a> Int J Pharm. 523 (2), 545-555 (2017).</li>
<li>Carozzi, V. A., Salio, C., Rodriguez Menendez, V., Ciglieri, E., Ferrini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((2d+vs+3d+morphological+analysis+of+dorsal+root+ganglia+in+health+and+painful+neuropathy%5BTitle%5D)+AND+%22Eur+J+Histochem%22%5BJournal%5D)">2d vs 3d morphological analysis of dorsal root ganglia in health and painful neuropathy.</a> Eur J Histochem. 65 (Suppl 1), 3276(2021).</li>
<li>Smith, P. R., Meyer, A., Loerch, S., Campbell, Z. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+the+isolation+and+culture+of+mouse+dorsal+root+ganglion+neurons+for+imaging+applications%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">Protocol for the isolation and culture of mouse dorsal root ganglion neurons for imaging applications.</a> STAR Protocols. 4 (4), (2023).</li>
<li>Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+roles+for+the+schwann+cell%3A+Ensheathment%2C+myelination%2C+trophism+and+regeneration+-+pubmed%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">Expanding roles for the schwann cell: Ensheathment, myelination, trophism and regeneration - pubmed.</a> Curr Opin Neurobiol. 3 (5), 805-809 (1993).</li>
<li>Chen, Z., Fang, Y., Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Important+cells+and+factors+from+tumor+microenvironment+participated+in+perineural+invasion%5BTitle%5D)+AND+%22Cancers%22%5BJournal%5D)">Important cells and factors from tumor microenvironment participated in perineural invasion.</a> Cancers. 15 (5), 1360(2023).</li>
<li>Jang, K., Garraway, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+dorsal+root+ganglia+and+primary+sensory+neuron+plasticity+mediating+inflammatory+and+chronic+neuropathic+pain%5BTitle%5D)+AND+%22Neurobiol+Pain%22%5BJournal%5D)">A review of dorsal root ganglia and primary sensory neuron plasticity mediating inflammatory and chronic neuropathic pain.</a> Neurobiol Pain. 15 (100151), (2024).</li>
<li>Hanani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+changes+in+neurons+and+satellite+glial+cells+in+mouse+dorsal+root+ganglia%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Age-related changes in neurons and satellite glial cells in mouse dorsal root ganglia.</a> Int J Mol Sci. 24 (3), 2677(2023).</li>
<li>Martinelli, C., Sartori, P., Ledda, M., Pannese, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+quantitative+changes+in+mitochondria+of+satellite+cell+sheaths+enveloping+spinal+ganglion+neurons+in+the+rabbit%5BTitle%5D)+AND+%22Brain+Res+Bull%22%5BJournal%5D)">Age-related quantitative changes in mitochondria of satellite cell sheaths enveloping spinal ganglion neurons in the rabbit.</a> Brain Res Bull. 61 (2), 147-151 (2003).</li>
<li>Niwa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+age-dependent+trophic+responses+of+nodose%2C+sensory%2C+and+sympathetic+neurons+to+neurotrophins+and+gdnf%3A+Potencies+for+neurite+extension+in+explant+culture%5BTitle%5D)+AND+%22Neurochem+Res%22%5BJournal%5D)">Differential age-dependent trophic responses of nodose, sensory, and sympathetic neurons to neurotrophins and gdnf: Potencies for neurite extension in explant culture.</a> Neurochem Res. 27 (6), 485-496 (2002).</li>
<li>Adler, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-dependent+differential+regulation+of+sensory+neuropeptides+by+glial+cell+line-derived+neurotrophic+factor%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">Age-dependent differential regulation of sensory neuropeptides by glial cell line-derived neurotrophic factor.</a> J Neurochem. 71 (1), 170-177 (1998).</li>
<li>Podratz, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotoxicity+to+drg+neurons+varies+between+rodent+strains+treated+with+cisplatin+and+bortezomib%5BTitle%5D)+AND+%22J+Neurol+Sci%22%5BJournal%5D)">Neurotoxicity to drg neurons varies between rodent strains treated with cisplatin and bortezomib.</a> J Neurol Sci. 362, 131-135 (2015).</li>
<li>Lawson, S. N., Biscoe, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+mouse+dorsal+root+ganglia%3A+An+autoradiographic+and+quantitative+study%5BTitle%5D)+AND+%22J+Neurocytol%22%5BJournal%5D)">Development of mouse dorsal root ganglia: An autoradiographic and quantitative study.</a> J Neurocytol. 8 (3), 265-274 (1979).</li>
<li>Monje, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roadmap+for+the+emerging+field+of+cancer+neuroscience%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Roadmap for the emerging field of cancer neuroscience.</a> Cell. 181 (2), 219-222 (2020).</li>
<li>Silverman, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer-associated+neurogenesis+and+nerve-cancer+cross-talk%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Cancer-associated neurogenesis and nerve-cancer cross-talk.</a> Cancer Res. 81 (6), 1431-1440 (2021).</li>
<li>Vermeer, P. D., Restaino, A. C., Barr, J. L., Yaniv, D., Amit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nerves+at+play%3A+The+peripheral+nervous+system+in+extracranial+malignancies%5BTitle%5D)+AND+%22Cancer+Discov%22%5BJournal%5D)">Nerves at play: The peripheral nervous system in extracranial malignancies.</a> Cancer Discov. 15 (1), 52-68 (2025).</li>
</ol>

The authors have no conflicts of interest to disclose.

The protocol presented here describes improved methods to establish 2D and 3D DRG primary nerve cultures and their application in co-culture with cancer cells. This protocol advances on previous methods by establishing the two models in parallel. As 2D and 3D models are established at the same time, using primary nerves isolated from the same animals, interanimal and interexperimental variation is minimized, and data from both models can be more easily compared or integrated facilitating complementary observations of cel...

Tumor dissemination, through local invasion and regional or distant metastasis, is the primary cause of cancer-related death. Although research has focused on vascular or lymphatic routes of spread, neural invasion of tumor cells is frequently observed in many solid tumors, including pancreatic1,2, prostate3, head and neck4, breast5, and colorectal6 cancers. In recent years, numerous studies have highlighted the emerging role of nerves in promoting tumor growth and progression, and as routes of dissemination, driving metastasis development7,8. In this context, cancer neuroscience has emerged as a prominent research field, offering new perspectives in oncology by elucidating the interactions between tumor cells and the fundamental components of the peripheral nervous system (PNS) that may contribute to these processes8,9.
The PNS consists of both peripheral nerves and ganglia, which are heterogeneous structures composed of diverse cell types, including neurons of varying sizes and functions, Schwann and other glial cells, fibroblasts, macrophages, mast cells, melanocytes, and vascular endothelial cells that form the local vasculature10. The PNS is a critical component of the tumor microenvironment (TME), and reciprocal communication between cancer cells and nerves, either directly through physical contact or indirectly through the release of neurotrophic factors, neurotransmitters, growth factors, and other signals, can facilitate neural invasion and promote more aggressive phenotypes8.
In vitro models can be used to characterize the processes that contribute to nerve maintenance and repair and are increasingly being used to examine cellular interactions between nerves and cancer cells11,12. A frequently used in vitro model involves extracted mouse dorsal root ganglia (DRG) co-cultured with tumor cells11. These models often use two-dimensional (2D) monolayer culture of dissociated DRG cells to study neuronal biology in vitro and have been used to model cellular interactions in the PNS13,14. However, 2D cultures lack the complexity of the nerve microenvironment and poorly represent its in vivo architecture. Three-dimensional (3D) whole mounts of peripheral neurons or ganglia offer an alternative that more effectively recapitulates peripheral nerve physiology to represent nerve-environment interactions more holistically15,16,17,18. However, the heterogeneity of this model limits the ability to visualize or quantify cell-cell interactions, and these models are more effective for observing overall peripheral nerve responses than for exploring specific cellular interactions and mechanistic responses to stimuli. Thus, it is clear that dissociated and whole mount neuronal cultures are complementary and that evaluation of 2D and 3D models in parallel is optimal to assess both overall effects and individual cell responses to stimuli.
This protocol describes methods for generating complementary dissociated (2D) and whole-mount (3D) nerve cultures that can be used to evaluate nerve and cancer cell interactions at both cellular and tissue levels, providing a more comprehensive view of these interactions than methods using only one type of culture1,13. These models can be used to visualize and assess cellular processes, including nerve cell morphology and neurite outgrowth, as well as tumor cell responses to nerve-secreted chemotactic agents, such as neurotrophic factors, and crosstalk between sensory neurons and tumor cells. It also allows exploration of potential interactions between tumor cells and other components of the neural microenvironment, such as immune cells, fibroblasts, and glia. We highlight the application of these models to study nerve-cancer cell interactions over time, visualized by live cell staining or immunofluorescence, as examples of approaches to assess or quantify cell interactions and responses. Together, the results generated from the use of these methods can further our understanding of how the nervous system promotes the growth and spread of cancer, helping us to develop strategies to target these mechanisms.

<table><tbody><tr><td>&lt;strong&gt;Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer</td><td>Progene</td><td>71-229483-ULT</td><td>Used for filtering cell suspension</td></tr><tr><td>8-well glass bottom slides</td><td>IBidi, Munich, Germany</td><td>80807</td><td></td></tr><tr><td>Dumont #5 - Fine Forceps</td><td>Fine Science Tools, Foster City, USA</td><td>11254-20</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools, Foster City, USA</td><td>11252-20</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools, Foster City, USA</td><td>91460-11</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Pattern Forceps</td><td>Fine Science Tools, Foster City, USA</td><td>91121-12</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Spring Scissors</td><td>Fine Science Tools, Foster City, USA</td><td>91500-09</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1x Phosphate buffered saline</td><td>Sigma, St. Louis, USA</td><td>D8662-500ML</td><td>Used for dissolving enzymes</td></tr><tr><td>B-27 Supplement (50x)</td><td>Thermo Fisher Scientific, Massachusetts, USA</td><td>17504044</td><td>Used at 2% concentration in F12 media</td></tr><tr><td>Bovine Type I Collagen</td><td>Corning Incorporated, New York, USA</td><td>354231</td><td>Used for coating plates, Working solution 50 &amp;micro;g/mL in 0.01 M HCl</td></tr><tr><td>Collagenase Type IV</td><td>Sigma-Aldrich, St. Louis, USA</td><td>C4-BIOC</td><td>Dissolve in PBS at 1.25 mg/mL, aliquot, store at -20 &amp;deg;C</td></tr><tr><td>Dispase II</td><td>Roche, Basel, Switzerland</td><td>4942078001</td><td>Dissolve in PBS at 2.5 mg/mL, aliquot, store at -20 &amp;deg;C</td></tr><tr><td>DNase I</td><td>Roche, Basel, Switzerland.</td><td>11284932001</td><td>Added at final concentration of&amp;nbsp; 0.2 mg/mL during dissociation</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>F1051</td><td>Used at 10% concentration in F12 media</td></tr><tr><td>HBSS (Hank&#039;s Balanced Salt Solution)</td><td>Sigma, St. Louis, USA</td><td>H6648-500ML</td><td>Prepared with 1% Penicillin-Streptomycin</td></tr><tr><td>Matrigel Growth Factor Reduced</td><td>Corning Incorporated, New York, USA</td><td>356231</td><td>Thaw at 4 &amp;deg;C overnight, keep on ice</td></tr><tr><td>Nutrient Mixture F-12 Ham</td><td>Sigma-Aldrich, St. Louis, USA</td><td>N6658-500ML</td><td>Basal medium for preparation of supplemented culture media</td></tr><tr><td>Papain</td><td>Roche, Basel, Switzerland.</td><td>10108014001</td><td>Dissolve in PBS at 30 U/mL, aliquot, store at -20 &amp;deg;C</td></tr><tr><td>Penicillin-Streptomycin (100x)</td><td>Sigma-Aldrich, St. Louis, USA</td><td>P4333-100ML</td><td>Used at 1% concentration in media</td></tr><tr><td>Trypan Blue Solution</td><td>Sigma-Aldrich, St. Louis, USA</td><td>T8154-100ML</td><td>To assess cell viability</td></tr><tr><td>TrypLE Express</td><td>Thermo Fisher Scientific, Massachusetts, USA</td><td>12605-028</td><td>For harvesting cancer cells</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;beta;-Tubulin III antibody</td><td>Abcam (Cambridge, UK)</td><td>AB15568</td><td>Dilution 1:200</td></tr><tr><td>Alexa Fluor 488</td><td>Thermo Fisher Scientific, Massachusetts, USA</td><td>A-11008</td><td>Dilution 1:2000</td></tr><tr><td>Alexa Fluor 594</td><td>Thermo Fisher Scientific, Massachusetts, USA</td><td>A-21442</td><td>Dilution 1:2000</td></tr><tr><td>Alexa Fluor 647</td><td>Thermo Fisher Scientific, Massachusetts, USA</td><td>A-21245</td><td>Dilution 1:2000</td></tr><tr><td>NeuN antibody</td><td>Sigma-Aldrich, St. Louis, USA</td><td>MAB377</td><td>Dilution 1:100</td></tr><tr><td>&lt;strong&gt;Fluorescent Dyes &amp;amp; Probes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ActinGreen 488 ReadyProbes Reagent (Phalloidin)</td><td>Thermo Fisher Scientific, Massachusetts, USA</td><td>R37110</td><td>Dilution 1:40</td></tr><tr><td>CellTrace Calcein Red-Orange</td><td>Thermo Fisher Scientific, Massachusetts, USA</td><td>C34851</td><td>Cell tracker used to visualize neurons under fluorescence microscope</td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher Scientific, Massachusetts, USA</td><td>H3570</td><td>Dilution 1:5000</td></tr></tbody></table>

establishing in vitro models of dorsal root ganglia culture: complementary approaches for investigating cancer-nerve crosstalk

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

The contribution of the nervous system to the tumor microenvironment and the importance of neural invasion as a route for cancer dissemination are being increasingly recognized. Interactions of cancer cells with neurons can promote their invasion around and into nerves, a feature of many cancers with poor clinical outcomes. In vitro models to study reciprocal interactions between neurons and cancer cells provide valuable tools for understanding cancer spread and identifying approaches to mitigate it. 
Here, we describe a protocol for murine dorsal root ganglia (DRG) isolation and the establishment of both whole mount and dissociated monolayer cultures that can be used to visualize neuron morphology and neurite outgrowth over time. Whole DRGs mounted in Matrigel preserve nerve architecture and responses to stimuli in a heterogeneous environment more similar to the in vivo nerve, while dissociated nerve cultures allow assessment of direct cell-cell interactions more closely. Once DRG cultures are established, cancer cells can be added to generate co-cultures that can be used to visualize changes in neurite outgrowth and nerve morphology in response to cancer cells. Growth or motility of cancer cells in response to nerve-derived signals over time or under conditions of growth stimulation or inhibition can be assessed, as well as visualizing the effects of direct contact between cancer cells and nerve extensions. 
As both co-culture models can be generated simultaneously, this protocol provides a more comprehensive view of the impact of cancer-neuron interactions and facilitates comparisons of treatment conditions and integration of information from the cellular level and whole ganglia. This protocol will facilitate the study of nerve-tumor interactions and can be used for a wide range of applications, including studies of cell signaling, drug screening, or study of the heterogeneity of the tumor-nerve environment and the mechanisms of tumor dissemination along nerves.

Our protocol describes the isolation of primary mouse DRG-derived cells and the establishment of two complementary culture models: dissociated DRG and whole-mounted DRG cultures, which allow us to investigate nerve-cancer cell interactions at different levels of complexity. A key advantage of this protocol is that both types of culture can be generated using DRGs isolated from the same animal, which minimizes inter-animal and inter-experimental variability and allows the information gener...

This protocol describes methods for establishing whole mount and dissociated cultures of mouse Dorsal Root Ganglia (DRG) and their use to assess tumor-nerve interactions.

The contribution of the nervous system to the tumor microenvironment and the importance of neural invasion as a route for cancer dissemination are being ...

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Research

JoVE Journal

Cancer Research

In this video, we demonstrate the procedure for developing an in vitro perineural cancer cell invasion model by co-culturing mouse dorsal root ganglion and neurotropic cancer cells. The model can be utilized to conduct cancer assays, producing rapid and reproducible results.

Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model to Study Perineural Cancer Cell Invasion

In this video, we demonstrate an in vitro method for co-culturing DRG and cancer cells to study neurotropism and neural invasion.

Modeling Cancerous Neural Invasion In Vitro: A Method to Co-culture Dorsal Root Ganglia and Cancer Cells

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

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

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P0-GGFβ3 mouse model.

Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models

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

Learn to establish complementary 3D and 2D dorsal root ganglia cultures from a single mouse for studying cancer-nerve crosstalk. This protocol enables investigation of neuro-guided tumor migration and therapeutic strategies.

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

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