Apr 06, 2026
Enzymatic hDRG Dissociation (room temperature, up to 2 digestion rounds)
- Felipe Espinosa Becerra1,
- Muhammad Saad Yousuf2,
- Lucy He2,
- Theodore Price2
- 1University of Texas at Dallas;
- 2Department of Neuroscience, School of Behavioral and Brain Sciences, University of Texas at Dallas
Protocol Citation: Felipe Espinosa Becerra, Muhammad Saad Yousuf, Lucy He, Theodore Price 2026. Enzymatic hDRG Dissociation (room temperature, up to 2 digestion rounds). protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbrk7ylpk/v1
Manuscript citation:
Franco-Enzástiga Ú, Natarajan K, Espinosa F, Granja-Vazquez R, Mydugolam H, Price TJ. Type I IFNs enhance human dorsal root ganglion nociceptor excitability and induce TRPV1 sensitization. JCI Insight. 2025 Sep 2;10(19):e194987. doi: 10.1172/jci.insight.194987. PMID: 40892477; PMCID: PMC12513479.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 30, 2025
Last Modified: May 18, 2026
Protocol Integer ID: 119309
Keywords: hDRG dissociation, room temperature, Stemxyme, 2 digestion rounds, enzymatic hdrg dissociation, fresh enzymatic solution in dissociation round, human dorsal root ganglion, significant proportion of neuron, larger proportion of neuron, neuron, digested tissue, hdrg, dissociation, fresh enzymatic solution, dissociation round, cell, digestion
Abstract
This protocol is to enzymatically dissociate and isolate human dorsal root ganglion (hDRG) neurons. The dissociation is done at room temperature (RT) where tissue is exposed to up to 2 dissociation rounds to have a better control over the extent of the digestion.
Round 1 includes cells already freed from tissue and with survival rates typically above 90% (vital staining with Trypan Blue). In this round, a significant proportion of neurons are surrounded by SGCs.
To be able to recover cells still embedded in incompletely digested tissue, it is re-exposed to fresh enzymatic solution in dissociation round 2. A larger proportion of neurons on round 2 are freed from SGCs, however, they usually have slightly lower survival rates.
Find attached a table of solution recipes and reagents.
Attachments
Image Attribution
Publication License Mar-31-2026.pdf Guidelines
Human neuronal cultures should be handled with proper safety precautions while handling and disposing.
Materials
Please find reagents and recipes in the word document attached.
Part A: Coverslip preparation (1 to several days before dissociation)
20h 20m
Disinfect and sterilize coverslips (12 mm) in fume hood in small petri dish using 70% EtOH. Dip coverslip in 70% EtOH, move to a 35 mm Petri dish - lean the coverslip on the side of the lid for the EtOH to dry. Can add 4-5 coverslips per dish. Airdry for a few minutes. For sterilization, leave them in the cell culture hood exposed to UV light for 20-30 minutes on each side.
Optional. For electrophysiology, use a cell culture insert (from IBIDI, Cat. # 80209). The insert comes with adhesive. Alternatively, glue a glass (cloning) cylinder (Fisher Scientific Cat. # 09-552-21) per coverslip. Use Loctite Glass Glue only (other superglues detach easily after several days in solution). Use small amounts along the edge in contact with the coverslip. To keep as many cells attached as possible, either type of insert is taken off the coverslip once it is placed inside the electrophysiology chamber ready for recordings.
Silicone Cell Culture Inserts and Cloning Glass Cylinders. Left. (Top) A two-well silicone cell culture insert. (Bottom) To save neurons for standard manual electrophysiology, the cell culture area inside the insert is reduced by cutting it first in half (semi-horizontal dotted red lines) and next doing 2 additional cuts (semi-vertical dotted red lines). This makes 2 L-shaped ‘walls’ (right-angled line) and 2 U-shaped walls (blue lines) per insert. Center. Four inserts in top of sterilized coverslips. The L-shaped wall (red) is used to contain plated neurons inside the U-shaped insert (blue). Right. Cloning glass cylinders glued to coverslips inside a Petri dish.
20m
Pre-treat coverslips with Poly-D-Lysine (PDL, >300,000 MW). Add 20-30 μl of PDL 100 μg/ml to each insert/coverslip. Incubate at 4°C overnight, or for 2-hrs at 37°C. Aspirate the PDL and rinse insert/coverslip 2-3 times with sterile water. Allow coverslips to air dry inside the biosafety cabinet. Cover Petri dish with parafilm and store at 4°C. (use within 1 month).
20h
Part B: Two-step DRG dissection, dissociation and cell plating
6h 35m
Prepare dissociation solution, BSA, culture media:
a. Dissociation solution. Prepare 1 mg/ml Stemxzyme I + 0.1 mg/ml DNAse I in 12 ml/DRG dissociation solution.
Stock solutions in HBSS: Stemxzyme I: 10 mg/ml (0.1 ml per ml of dissociation solution).
DNAse I: 4 mg/ml (25μl per ml of dissociation solution).
NOTE: Preliminary assessment indicates that 1 ml of the dissociation solution is good for 10-25 mg of cleaned DRG. The younger the donor, the larger the amount of DRG mass per ml. About 65-70% of the total solution is used from start, leaving 30-35% as fresh solution for round 2 (see next and step 9).
b. Prepare BSA 12%. Prepare 20-50ml of BSA 12% in HBSS (120mg/ml). Shake gently to dissolve (do not vortex). Then filter sterilize (we use Steriflip from Fischer Scientific, Cat. #SCGP00525) and pour 2-3 ml in 2-4 15 ml conical tubes. Keep at room temperature.
c. Prepare Culture Media. To Brain Phys add: 1% Pen Strep, 2% SM1, 1% N2, 1% GlutaMax, 10-20 ng/ml
hNGF (stock 100 μg/ml) and 5-10 ng/ml of GDNF (stock 100 μg/ml).
NOTE: follow recipes on the attached document for these and other solutions in this protocol.
10m
Dissection (chilling temperature). Place DRGs on a Petri dish with Sylgard filled with sterile NMDG-aCSF, (use painted lid underneath for contrast). To ease dissection procedure, afix DRGs with several 25-gauge needles penetrating the Sylgard. Clean up the DRG by removing excess fat, connective tissue, meninges, epineurium and nerve/roots in NMDG-aCSF (see flow diagram on step 16) (Valtcheva et al., 2016). Weigh the DRG (s).
Petri dishes with Sylgard. For better contrast during dissection, lids were painted black and placed underneath the Petri dish.
Note: the dissection solution can be prepared in the previous step and final volume adjusted after weighing DRGs. Alternatively, prepare the exact amount of dissection solution after DRGs are weighed.
30m
Mince (chilling temperature). In a 35 mm Petri dish filled with sterile NMDG-aCSF, cut the DRGs into small 0.4-0.5 mm pieces (can also do larger 1-2 mm pieces). Afterwards, transfer DRG pieces to the dissociation solution in conical tube 1.
Physical and Enzymatic Dissociation (at room temperature). Parafilm and place the conical tube in a shaker. We use a nutator shaker at 60-70 RPM for 90-180 min till the tissue starts loosening (you will see some tangles of fiber-like threads separate from the tissue chunks).
Note: The total digestion time in this first round depends on the size of the DRG pieces, and on the age of the donor. The larger the pieces and the older the donor, the longer the dissociation time.
3h
Trituration. Using wide-bore fire-polished Pasteur pipettes, gently triturate by doing 10-15 strokes per Pasteur pipette tip size. Adjust the tip of the glass pipette (big to small). Go to the next smaller size once it becomes easy to suck into and release tissue from pipette (when triturating, avoid creating bubbles to decrease cell damage).
Note: For this first step, we use 3 pipette tip sizes of diameters of about 2, 1.5 and 1 mm. Increase these
diameters if using larger DRG pieces. For the smaller size pipette, first strokes will be slow because
relatively large tangles will be present.
40m
Start round 2 dissociation. Pull undigested tissue with the paster pipette and place it in tube 2 (round 2) with the remaining fresh dissociation solution to continue further enzymatic digestion at room temperature for 45-90 more minutes while continuing next steps in the first round.
5m
Check round 1 neurons for SGCs coverage. Drop 50-100 μl of the cell suspension from round 1 on a Petri dish and place a 10-12 mm coverslip on top, wait 1-2 min for neurons to sediment and inspect cells under the microscope. Check how many DRG neurons are covered by glia. If a significant number (>10-20%) are devoid from SGCs, go to next step. However, if most cells are still completely covered by glia, prepare and add 2-3 ml of fresh digestion solution and subject cells to re-digestion, but now at 37°C for 10-20 more minutes.
To visualize neurons and determine if SGCs are still covering them, 100 μl of cell suspension are placed on a Petri dish (left) as well a coverslip on top of it (center). Right. The picture shows two hDRG neurons covered with SGCs (black arrows), as well as 1 hDRG neuron that has a significant amount of its surface free from SGCs and suitable for patch clamp (yellow arrow).
5m
BSA gradient centrifugation. Carefully add 2-3 ml of the cell suspension on top of the 12% BSA (avoid disrupting the BSA gradient). Spin down (500g for 9 min at RT). Use slow acceleration and slow deceleration settings for the centrifugation, otherwise, the BSA gradient will be disrupted allowing lots of debris to the pellet.
Optional. If debris is abundant, pass cell suspension through a 100 μm cell strainer before centrifugation. Occasionally, more than one cell strainer will be needed if the strainers easily clog with debris.
15m
Recover neurons and determine neuronal count and survival rate (Trypan Blue 4%). Carefully discard supernatant without disturbing the pellet. Add 100-200 μl of Brain Phys media and resuspend cells. To a 2-3 μl drop of Trypan Blue add 2-3 μl of re-suspended cells, mix and incubate 1 min. Cover with a 10-12 mm coverslip and count stained neurons vs non stained. Total neuronal count for round 1 = ((stained + non stained neurons) / microliters of cell suspension added) * Brain Phys medium volume added. Cell cytoplasm stains with Trypan Blue when the plasma membrane is damaged (this represent dead cells). Survival rate = non-stained neurons/total X 100. On round 1 it is common to get survival rates above 90%.
15m
Cell Stock round 1. Dilute cells to the adequate cell-stock concentration for your experiments. We usually suspend cells to a concentration of 5 cell/μl of culture media and store them in a tube (Eppendorf or screw-cap tube) to be stored at RT until plating them. Replace half of culture media in tube twice a week. In our hands, most RT stored cells remain viable for plating at least for 2 weeks.
5m
Plate Cells. Plate 2-4 coverslips per day (see part A for coverslip preparation). When using inserts or cloning glass cylinders, the Petri dish can be pre-flooded with 3 ml of culture media. Otherwise, add 10-20 μl of cell suspension to each coverslip and allow 1-2 hrs for the cells to adhere strongly to the coverslip. Carefully add 1 ml of media at a time (avoid disrupting cells as much as possible).
Optional. Pre-coat coverslips with 20-30 μl of laminin in culture media (1
Note: If debris is still abundant, debris will sediment on top of the neurons covering them and precluding physiology experiments. Fortunately, most debris can be discarded by resuspending the cells and letting neurons sediment for 3-5 minutes (most debris will sediment slowly many minutes after neurons). Discard 60-90% of the supernatant. To keep the desired cell concentration, replace the same discarded volume with fresh culture media.
15m
Culture Cells (1-10 days or more). Use regular cell culture conditions (37°C - 5% CO2). Replace half the media volume with fresh solution every other day. Perform physiology experiments 1-10 days after plating or as planned.
Second round. Repeat steps 7 and 9-14 for second round. Compared to round 1, debris is usually higher and survival rate is lower in round 2.
1h 15m
Flow Diagram of hDRG Dissociation at Room Temperature.
After collecting the hDRGs, they are cleaned at chilling temperature by removing excess fat, connective tissue, meninges, epineurium and nerve/roots and minced in small pieces of 0.4- 0.5 mm (1, 2). Minced DRG pieces are subjected to enzymatic and mechanical dissociation at room temperature, first by agitation in a shaker and later by trituration with Paster pipettes (3.I). Undigested tissue is recovered with the Paster pipette and placed in a new tube with fresh enzyme (3.II). After adequate tissue digestion, cells are recovered by centrifugation in a BSA gradient (4.I, 4.II). Pellet is
resuspended in culture media, cells are counted and survival rate determined (5.I, 5.II). Cells are plated and subsequent physiological experiments are done after a few days in culture (6.I, 6.II).
Protocol references
Franco-Enzástiga Ú, Natarajan K, Espinosa F, Granja-Vazquez R, Mydugolam H, Price TJ. Type I IFNs enhance human dorsal root ganglion nociceptor excitability and induce TRPV1 sensitization. JCI Insight. 2025 Sep 2;10(19):e194987. doi: 10.1172/jci.insight.194987. PMID: 40892477; PMCID: PMC12513479.
Valtcheva, M., Copits, B., Davidson, S. et al. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures. Nat Protoc 11, 1877–1888 (2016). https://doi.org/10.1038/nprot.2016.111
Acknowledgements
We thank Anna Cervantes, Geoffrey Funk, and Peter Horton at the Southwest Transplant Alliance. We are grateful to the organ donors and their families for their donations.