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 respo....

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
   .......

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)