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

Summary

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.

Abstract

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.

Introduction

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 stimuli^4,5. 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 therapeutics^8,9. 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 platforms^13,14, 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' 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 neurons^17,18,19.

Protocol

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

1. Computer-aided design and 3D printing of the multi-compartment device
   NOTE: The MC device consists of three compartments for the isolation of DRG cell bodies and neurites (Figure 2A). The MC device is not commercially available; however, the STL file is provided herein (Supplementary Coding File 1) to enable 3D printing. Printing the MC takes one (1) day to complete.
2. Upload the STL file of the MC to a Resin 3D printer using compatible software.
   NOTE: The material ideal for printing is a high-temperature resin. A key advantage of using high-temperature resin is it is biocompatible and can be autoclaved and reused. A Digital light processing (DLP) 3D printer or other 3D printers could also be used to print the device.
3. Transfer the printed devices into a washing solution for 30 min in isopropanol (>96%) to remove residual resin from the surface of the printed MC device.
4. Post-cure the devices at 80 °C for 120 min using a commercially available curing agent (see Table of Materials). The curing agent enhances the mechanical properties of the printed MC device by exposing it to temperature and UV.
5. Cure the MC devices thermally in an oven at 160 °C for 180 min.
6. Sterilize the devices using an autoclave for 45 min on a gravity cycle.

2. Hydrogel preparation

1. Synthesize methacrylated hyaluronic acid (MAHA, 85%-115% methacrylation) and characterize using a protocol described by Seidlits et al.²⁰.
   NOTE: To support the growth of DRGs, triple hydrogels composed of methacrylated hyaluronic acid, type I collagen, and laminin are commonly used. Findings indicate that DRGs commonly grow in MAHA, ranging from 1.25-2.5 mg/mL and collagen from 2.0-4.5 mg/mL. For laminin, it is found that 0.75 mg/mL is enough to ensure robust growth of DRGs. Herein, methods to create a triple gel with 1.25 mg/mL MAHA, 4.5 mg/mL collagen, and 0.75 mg/mL laminin are detailed. Hydrogel preparation should be done in a laminar flow cabinet to provide an aseptic workspace.

3. Photoinitiator solution preparation

NOTE: A photoinitiator is necessary to crosslink the MAHA under UV light. It is commonly used at percentages from 0.3% to 0.6%.

1. Prepare the photoinitiator solution 3 days prior to the DRG harvest.
2. Pre-heat the sonicating water bath to 37 °C before preparing the photoinitiator solution.
3. Working with the sterile technique in a flow hood, prepare 23.125 mg/mL sodium bicarbonate (NaHCO₃) solution by dissolving NaHCO₃ in 5x Dulbecco's Modified Eagle Medium (DMEM)-HEPES solution.
4. In a glass vial, mix 0.6% weight/volume (w/v) photoinitiator in a 20% vol/vol 1x sterile phosphate-buffered saline (PBS) and 80% vol/vol 5X DMEM/HEPES solution.
5. To protect from light, wrap the glass vial in aluminum foil. However, ensure to remove the aluminum foil before transferring the vial to the sonicating water bath heated to 37 °C with sonication between 30-40 min.
   NOTE: Photoinitiator solution must be protected from light because it can break down into radicals and start the polymerization process when exposed to light²¹.
6. Sterile filter the solution with a 0.22 µm syringe filter into a new sterile glass vial.

4. Methacrylated hyaluronic acid dissolution

1. Dissolve the methacrylated hyaluronic acid (prepared in step 2) on the same day the photoinitiator solution is prepared (step 3).
2. Place the frozen lyophilized MAHA in a desiccator for 15 min to come to room temperature.
3. Working in a laminar flow hood and using the sterile weighing technique, weigh the lyophilized MAHA in a glass vial.
4. Add the necessary volume of filtered photoinitiator solution to MAHA to come to the desired MAHA concentration. The common final gel concentration is 1.25 mg/mL MAHA and 4.5 mg/mL collagen.
5. Seal the cap with laboratory sealing film, wrap the glass vial with aluminum foil, and place it on an orbital shaker plate at room temperature until fully dissolved (usually 3 days). Occasionally, vortex the solution to break apart large clumps of MAHA to ensure uniform dissolution of MAHA.

5. Device assembly

1. Place the MC device into a 48-well plate a day before the DRG harvest.
2. Apply a thin line of silicone gel in the groove beneath the MC device (Figure 2B) using a 3 mL syringe. This will help the device to securely attach to the well plate.
3. With the help of #3 forceps, gently place the MC device into the well of a 48-well plate, as shown in Figure 2C.
   NOTE: The size of the device can be modified to suit the intended application, and a plate of a different size can be used if desired. The devices can be reused. To reuse, the hydrogel must be gently washed out from all tunnels, and the loading groove beneath the device must be used with 70% ethanol with gentle agitation. After this is complete, devices can be re-autoclaved to sterilize prior to use.

6. Animal preparation

1. Autoclave all the necessary dissection tools prior to the start of harvest.
2. Obtain an adult male or female Sprague Dawley rat aged 12-20 weeks. The sex of the rat does not impact DRG growth.
3. Euthanize the rat according to the approved IACUC protocol using CO₂ overdose as the primary method of euthanasia and bilateral pneumothorax puncture as a secondary method of euthanasia according to American Veterinary Medical Association Guidelines²².
4. Place the rat on a sterile underpad on a dissecting table with the ventral side down. Spray the fur down with 70% ethanol.

7. Dorsal root ganglia (DRG) harvest

1. Prepare trimming media containing 86% Neurobasal A media, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PS), 1% Glutamine, and 2% B27 plus 50x and transfer into a 24-well plate. This media and well-plate will be used for temporary storage of harvested DRGs prior to embedding them in the hydrogel.
2. Wear a lab coat, surgical mask, bonnet, safety glasses, and gloves.
3. Using large blunt-nose scissors, cut through the skin at the base of the cervical spine and then down the length of the spine.
4. To further drain blood away from the spinal canal, cut underneath the shoulder blades and down through the muscle to sever major blood vessels.
5. Use large, sharp-nose scissors to cut the muscle away from the spine. Clear as much muscle as possible to visualize the spine.
6. Remove the dorsal and lateral segments of the vertebrae. Use a straight Rongeur and sharp-nosed scissors to remove the spinous and transverse processes of the vertebrae, starting at the cervical end and moving toward the lumbar end.
7. Remove the spinal cord. Use small sharp-nose scissors to carefully cut through the nerve roots connected to the DRGs along both sides of the spinal cord. Cut the cervical end of the spinal cord and carefully lift out of the spinal canal gradually, cutting through any remaining nerve roots connected to the DRGs.
   NOTE: Fine motor skills are essential to ensure the DRG body is not severed in the process.
8. To expose DRGs, use the curved Rongeur to remove more of the lateral parts of the vertebrae. Pull outward from the midline, alternating sides. Be careful not to cut through the DRG body since it may cause damage to the cell bodies and axons.
9. Using the #3 forceps and straight-edge spring scissors, remove DRGs in between each level of the vertebrae. Grip the spinal nerve roots from the DRG, carefully pull them out of the pocket, and cut through the other end of the spinal nerve. Be careful not to grip the DRG body directly.
10. Place the DRGs in wells of a 24-well plate (containing trimming media, step 7.1) on ice.
11. After collecting all DRGs, clean up the workspace, place the carcass in an autoclave bag, and store/dispose of it appropriately according to IACUC protocol.

8. DRG trimming and cutting

1. Fill a 60 mm Petri dish on a clean bench with trimming media (step 7.1). Set up the dissecting scope and glass bead sterilizer and place an autoclaved toolbox with trimming tools (#3 forceps and straight-edge spring scissors) by the scope.
2. Under the dissecting scope, remove excess tissue around the DRG and trim the nerve roots close to the DRG body (typically slightly darker than the surrounding nerves).
3. Fill a second 60 mm Petri dish with fresh media. Under the dissecting scope, cut the trimmed DRGs to about 0.5 mm (or between 0.5 mm and 0.8 mm). A ruler is placed under the Petri dish during cutting to have the correct estimated size of cut DRGs.
4. Transfer cut DRGs into a fresh 24-well plate with the media on ice.
   NOTE: DRG trimming, cutting, and embedding should be done in a laminar flow cabinet to provide an aseptic workspace. In the absence of a laminar flow cabinet, aseptic techniques must be observed, with minimum appropriate personal protective equipment (PPE) recommended (lab coat, masks, goggles, and gloves) and all tools sterilized throughout the process to mitigate potential contamination.

9. Collagen neutralization

1. Neutralize the collagen on the same day as the DRG harvest after trimming and cutting the DRGs.
2. Working on ice, pipette ~8.16 % ultra-pure water, ~1.84% 1 M sodium hydroxide (NaOH), ~10% 10x PBS, and ~80% of collagen of the total volume of solution into a glass vial.
3. Mix slowly using a sterile low-retention pipette tip to pipette collagen (usually added last) into the mixture. Ensure to keep the pipette tip in the solution while mixing to avoid creating bubbles. Check the pH of the neutralized collagen using pH strips to be sure it is ~7.
   NOTE: Neutralized collagen can be kept on ice for 2-3 h without gelling; therefore, hydrogel fabrication should be carried out expeditiously once collagen is neutralized.

10. Hydrogel fabrication

1. Working on ice, pipette laminin (to a final concentration of 0.75 mg/mL) and 1x PBS into a glass vial.
2. Using sterile low retention pipette tips, pipette neutralized collagen and MAHA solution into the mixture to arrive at the desired concentration.
3. Mix slowly and check the pH of the resulting hydrogel (should be ~7).

11. DRG embedding

1. Perform multi-compartment embedding.
   1. Pipette 65.1 µL and 52.8 µL of the pre-mixed hydrogel into the soma and neurite compartments, respectively.
      NOTE: This assumes the use of MC devices in a 48-well plate. These volumes can be changed based on the size of the MC device used.
   2. Gently embed the cut DRGs into the soma compartment (Figure 2A), ensuring they do not scratch the bottom of the well plate. Place the DRG in the middle so the neurites can grow into the adjacent compartments through the tunnels.
   3. Thermal crosslink the hydrogel in the incubator at 37 °C for 30 min. While this occurs, turn on the UV lamp to warm up.
   4. After thermal crosslinking, UV crosslink the hydrogel for 90 s.
   5. Add DRG growth media to the hydrogel and DRG and perform a full media change after an hour to eliminate unreacted or remaining traces of photoinitiator that persist in the media.
   6. Perform a half media change every 3 days and monitor the growth of the DRGs by viewing under the microscope.
   7. After ~4 weeks, when DRG neurites begin to grow (Figure 3A), capture images with the fluorescent plate imager and quantify the length of neurites using FIJI (ImageJ).

12. Control DRG embedding

1. Pipette 250 µL of hydrogel in a 48-well plate (without MC).
2. Gently place the cut DRG in the middle of the well and halfway down into the hydrogel, ensuring it does not scratch the bottom of the well plate.
3. Thermal crosslink the hydrogel in the incubator at 37 °C for 30 min.
4. UV crosslink the hydrogel for 90 s.
5. Add DRG growth media to the hydrogel and DRG and perform a full media change after an hour.
6. Perform half media change every 3 days and monitor the growth of the DRG by viewing it under the microscope.
7. After ~4 weeks, when DRG neurites begin to grow (Figure 3B), capture images with the fluorescent plate imager.
   NOTE: Any brightfield microscope will work for imaging. An automated plate-based microscope makes this process faster. Control embedding in plain gels without the device is done to compare the growth of DRG neurites in multi-compartment. Once users validate that DRGs grow similarly in MC and control gels, they may not need to repeat control gels each time.

13. DRG imaging

1. Capture brightfield images at 4x magnification using a fluorescent plate imager at a range of focal distances to visualize the entire DRG and MC Device.
2. Adjust the brightness and contrast of the image to make it easier to view the neurites.
3. Save the image as a .tiff file.

14. DRG neurite quantification

1. Download and install FIJI (ImageJ).
2. Open ImageJ. Open the image file and ensure that the file is monochrome.
3. Enhance the contrast so that the smallest neurites are visible. Do this by opening the brightness and contrast option using the shortcut Ctrl+Shift+C.
4. Adjust the sliders of the controller as needed to visualize the neurites, and click on Apply for the desired brightness and contrast.
5. Open the neurite tracer by opening the Plugins menu, selecting Segmentation, and clicking on Simple Neurite Tracer.
6. Start a new trace by clicking on one end of the neurite right up against the DRG body.
7. Click another point along the neurite to add a trace until the neurite is traced end to end. Click on Finish Path after tracing the neurite.
8. Convert the traced length to real units and save the results by copying and pasting them into Excel.
9. Use the straight-line tool in FIJI to draw a line of the same length as the scale bar (not letting go of the end of the line) and look at the length value visible under the toolbar. The length value is used for the conversion.
10. Measure the length of six long neurites extending from both sides of the DRG (Figure 3C,D) and calculate the average length of these neurites to give a representative average length of the DRG, as shown in Figure 4.
    NOTE: Immunostaining has been tested on DRG explants in plain gels to show the presence of non-neuronal support cells⁷. DRGs are fixed in 4% paraformaldehyde (PFA) at room temperature, washed three times with 1x PBS for 15 min, and stored in 1x PBS at 4 °C until immunostaining.
11. For explants in MC devices, remove the MC device and follow the same process described above⁷.

Results

The present protocol described a technique to harvest and culture DRG from adult Sprague Dawley rats in a multi-compartment (MC) device. As shown 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...

Discussion

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 DRGs^23,24,25. Damage to the spinal cord limits blood supply, which affects DRGs and internal neurons²⁶

Materials

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