a technique for harvesting and dissociating dorsal root ganglia for neuronal cultures

A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures

To obtain the DRG neurons after harvesting the spinal cord, begin by removing any dorsal parts and then, use sterile and sharp surgical scissors to divide the spinal column in half along the longitudinal axis, exposing the cord tissue. Cut the spinal column into two smaller segments below the level of the rib cage, and then use fine forceps to gently remove all the cord tissue, taking care not to pull and remove the DRG roots, which appear as white filaments coming directly out of the canals.
Once all of the core tissue has been removed, use very fine forceps to pull the entire DRG root, reaching deep into the vertebral canals and taking care not to damage the roots of the ganglia. Place the DRG into a 60-square-millimeter Petri dish containing 3 to 4 milliliters of Ham&#39;s F-12 medium, supplemented with antibiotics. Then, under a dissecting microscope, use sterile forceps and a scalpel to clear away any excess nerve roots surrounding the ganglia to reduce satellite cells&#39; contamination.
Next, transfer the DRG into a 35-square-millimeter Petri dish containing 1.8 milliliters of fresh F-12 medium, and add 200 microliters of collagenase type-4 stock solution. Incubate the DRG for one hour, and then carefully aspirate the medium with a glass pipette, taking care not to aspirate or damage the DRG.
Repeat the collagenase step and wash the DRG with F-12 medium. After the second wash, add 1.8 milliliters of F12 medium and 200 microliters of trypsin to the cells, removing the trypsin and adding 1 milliliter of medium supplemented with 500 microliters of FBS to arrest the enzymatic reaction. Then, aspirate the medium and gently wash the DRG with F-12 medium three times to remove all traces of the serum.
Now, feed the cells with 2 milliliters of fresh F-12 medium and use a glass pipette to carefully transfer the cell suspension into a 15-milliliter tube. Pipette up and down 8 to 10 times to gently dissociate the neurons, and then allow the pellet to accumulate at the bottom of the tube. When the pellet is settled, transfer the supernatant into a new tube, and add 2 milliliters of fresh F12 medium to the pellet, repeating the mechanical dissociation and medium transfer until the suspension becomes homogeneous.
Then, triturate the cell suspension three to four times, followed with filtration of the resulting homogenized suspension through a 100-micron cell strainer into a new 50-milliliter tube. Centrifuge the cells in a 15-milliliter tube. While they are spinning, slowly pipette freshly prepared 15% bovine serum albumin down the inside of a 15-milliliter tube held at a 45-degree angle to create a gradual protein trail using the numbers on the tube as a reference for the forming track.
Then, aspirate the supernatant from the cells, saving the last 500 microliters for resuspending the pellet, and slowly dispense the cells along the protein trail into the new tube. After spinning down the cells again, resuspend the pellet in 1 milliliter of modified BS medium and seed the cells in a small volume of medium in a 24-well plate for two hours. When the cells have attached, add fresh BS medium supplemented with 50 nanograms per milliliter of nerve growth factor.

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Neuronal Culture Techniques

Primary Neuronal Culture

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Set-up
<ol>
	<li>Prior to starting tissue and cell harvest, check that all the tools are sterile. If necessary, autoclave a pair of sharp surgical scissors, a pair of very fine forceps, and a pair of fine standard forceps. Also, as appropriate, sterilize each substrate before cell seeding using UV sterilization, ethanol exposure, or steam sterilization.</li>
	<li>Preparation of media and stock solutions for neuron harvest and dissociation
	<ol>
		<li>Prepare Bottenstein and Sato&#39;s (BS) medium, by adding 1% penicillin-streptomycin (PS) and 1% N2 supplement to Ham&#39;s F12 medium. Calculate the final volume needed as 500 &#181;l per well (if using a 24-well plate).</li>
		<li>Prepare collagenase IV stocks in Ham&#39;s F12 medium at the concentration of 1.25% wt/v. Filter-sterilize the solution and store at -20 &#176;C as 200 &#181;l aliquots.</li>
		<li>Prepare bovine pancreatic trypsin stocks in Ham&#39;s F12 medium at the concentration of 2.5% wt/v, filter-sterilize and store at -20 &#176;C as 200 &#181;l aliquots.</li>
		<li>Prepare the nerve growth factor (NGF) stock solution at the concentration of 5 &#181;g/mL in a filter-sterilized 1 mg/ml fatty acid-free bovine serum albumin (BSA) solution in F12 medium and store at -20 &#176;C as 200 &#181;l aliquots. Do not filter the NGF after reconstitution.</li>
	</ol>
	</li>
	<li>In case no surface modification has been performed, coat coverslips/plates with poly-D-lysine (0.1 mg/ml for 15 min at room temperatre [RT]) and/or laminin (2-10 &#181;g/cm2 for 2 hr at 37 &#176;C) to support neuron attachment and neurite outgrowth as appropriate.</li>
	<li>Always warm up media in a water bath at 37 &#176;C before using.</li>
</ol>
2. Harvest and Dissociation of Dorsal Root Ganglia (DRG) Neurons
<ol>
	<li>Terminate the rat by cervical dislocation and decapitation. Shave the rat and lift the skin to expose the spinal column. Using sharp scissors, excise the spinal column taking extra care of the confined organs and blood vessels. Transfer the spinal column in a biological safety cabinet (class II) using a Petri dish and remove any dorsal part.</li>
	<li>Divide the spinal column in half along the longitudinal axis using sterile and sharp surgical scissors to expose the cord tissue. At this point, it is helpful to cut the spinal column in two smaller segments below the level of the rib cage, to make it easier to handle during the DRG neuron harvest. Using fine forceps, gently remove all the cord tissue, paying attention to not pull and remove the DRG roots. This way the DRG and roots will be exposed within the vertebral canals, still encased in the column. Observe DRG as white filaments coming out directly from the canals.</li>
	<li>Pull out the entire DRG root (not just the DRG) from the vertebral canals by using very fine forceps, going deep into the vertebral canals and paying attention not to damage the roots of the ganglia. Transfer the DRG into a small petri dish (60 mm2) containing 3-4 ml of Ham&#39;s F12 medium supplemented with 1% PS. If using different animals, use separate dishes.</li>
	<li>Under a dissecting microscope, clean the DRG of any excess of nerve roots surrounding the ganglia using sterile forceps and a scalpel to reduce glial cell contamination. Transfer the DRG into a small petri dish (35 mm2) with 1.8 ml of fresh F12 medium.</li>
	<li>Add 200 &#181;l of 1.25% wt/v collagenase type IV stock solution (final concentration of 0.125%) and incubate the DRG at 37 &#176;C, 5% CO2 for 1 hr. Carefully aspirate the medium with a glass pipette, paying attention not to aspirate or damage the DRG. Add fresh F12 medium supplemented with 0.125% wt/v collagenase type IV enzyme and incubate for 1 hr as previously described.</li>
	<li>Aspirate the medium and gently wash the DRG with F12 medium. Add then 1.8 ml of F12 medium and 200 &#181;l of trypsin (final concentration of 0.25% wt/v) and incubate at 37 &#176;C, 5% CO2 for 30 min.</li>
	<li>Remove trypsin and add 1 ml of F12 medium supplemented with 500 &#181;l of fetal bovine serum (FBS) to arrest the enzymatic reaction. Aspirate the medium and gently wash the DRG with F12 medium for three times to remove traces of serum.</li>
	<li>Add 2 ml of fresh F12 medium and carefully transfer the DRG with the medium into a 15 ml tube using a glass pipette. Gently dissociate the DRG neurons by pipetting up and down (about 8-10 times) with the glass pipette (plastic tips can be used as alternative).</li>
	<li>Allow the pellet to settle at the bottom of the tube and collect the medium in a new tube. Add 2 ml of fresh F12 medium to the tube containing the pellet and repeat the mechanical dissociation with the glass pipette. Repeat this step until the suspension becomes homogeneous (about 3-4 times) and collect all the dissociated DRG in the new tube. This method reduces the stress deriving from the mechanical dissociation and improves the viability of the neurons.</li>
	<li>Filter the resulting homogenized suspension into a new 15 ml tube using a 100 &#181;m cell strainer to remove un-dissociated neurons and other debris. At this stage, it might be convenient to first filter the cell suspension into a 50 ml tube and then transfer the solution into the smaller tube using a glass pipette. Centrifuge the suspension at 110 g for 5 min.</li>
	<li>Prepare a 15% BSA by adding 500 &#181;l of a 30% BSA solution to 500 &#181;l of F12 medium. Slowly pipette the solution down the wall of a 15 ml tube to create a gradual protein trail. At this stage, it is helpful to hold the tube at a 45&#176; angle and slowly release the BSA using the numbers on the falcon tube as a reference for the forming &#34;track&#34;.</li>
	<li>Aspirate the supernatant from step 2.10 leaving 500 &#181;l at the bottom of the tube and resuspend the cell pellet in the same medium. Slowly pipette the suspension along the protein trail previously prepared in step 2.11 (use the numbers on the tube as a reference) and centrifuge at 500 g for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 1 ml of modified BS medium. 
	NOTE: The volume to re-suspend the DRG neurons can be made up to the actual volume required, depending on the final cell concentration desired and the number of samples to be seeded. One animal will provide enough cells for a 24-well plate experiment.</li>
	<li>Incubate the seeded samples at 37 &#176;C, 5% CO2 for 2 hr to allow cell attachment and finally add fresh BS medium supplemented with 50 ng/ml of NGF.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 &#181;m cell strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td>70 &#956;m strainers (ref. 352350) can be used as alternative</td>
		</tr>
		<tr>
			<td>15 mL plastic tubes</td>
			<td>Sarstedt</td>
			<td>62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL plastic tubes</td>
			<td>Sarstedt</td>
			<td>62.547.004</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 flasks</td>
			<td>Corning</td>
			<td>BC301</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9205</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type IV</td>
			<td>Worthington Biochemical</td>
			<td>LS004188</td>
			<td>Note: this collagenase is only used to dissociate DRG explants</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (FBS)</td>
			<td>Biosera</td>
			<td>FB-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipettes</td>
			<td>Fisher Scientific</td>
			<td>FB50253</td>
			<td>Sharp material to be disposed accordingly</td>
		</tr>
		<tr>
			<td>Nutrient Mix F12 HAM</td>
			<td>Sigma</td>
			<td>N6658</td>
			<td>Warm at 37 &#176;C in a water bath unless specified</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Sigma</td>
			<td>H9394</td>
			<td>Warm at 37 &#176;C in a water bath unless specified</td>
		</tr>
		<tr>
			<td>N-2 supplement (100x)</td>
			<td>Invitrogen</td>
			<td>17502</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve Growth Factor 2.5s Protein, Mouse Submaxillary Glands (NGF)</td>
			<td>Millipore</td>
			<td>NC011</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (PS)</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (2x bovine pancreatic)</td>
			<td>Worthington Biochemical</td>
			<td>LS003703</td>
			<td>This is used for DRG dissociation</td>
		</tr>
	</tbody>
</table>

Source: de Luca, A. C., et al., <a href="https://app.jove.com/v/52543">Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration.</a> J. Vis. Exp. (2015).
This video demonstrates a method for harvesting neurons from the dorsal root ganglia (DRG). DRG from a rat&#39;s spinal column is isolated, treated with enzymes, and then mechanically dissociated to obtain a single-cell suspension. The neurons are separated from glial cells using density gradient centrifugation and cultured in a growth medium.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Set-up

	Prior to ...

Take a section of a rat&#39;s spinal column and divide it to remove the spinal cord.
Detach the dorsal root ganglia, or DRGs, a cluster of neurons surrounded by satellite glial cells, or SGCs.
Place the DRGs in a growth medium. Remove the nerve roots to reduce SGC contamination.
Treat with collagenase to degrade the tissue matrix and wash to remove the enzymes.
Introduce trypsin to disrupt intercellular connections. Add a serum containing proteins that deactivate trypsin.
Add the growth medium and mechanically dissociate the tissue. Filter the suspension to isolate single cells and centrifuge to remove tissue debris.
Resuspend the cells to layer them on a density gradient medium, and centrifuge.
Neurons with a higher density settle at the bottom, facilitating the separation of SGCs.
Resuspend the neurons in a culture medium. Transfer them onto a culture substrate-coated well, allowing cell attachment.
Supplement with nerve growth factors for neuronal survival.

43 次观看. 一种用于神经元培养的背根神经节收获和解离的技术

视频: 一种用于神经元培养的背根神经节收获和解离的技术 - 实验

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

科研

JoVE Journal

神经科学

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

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

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.

大鼠背根神经节外植体和雪旺细胞共培养中外周轴突的体外髓鞘形成

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

小鼠背根神经节冷冻切片

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of inflammatory and neuropathic pain, itch, as well as other peripheral neurological conditions. However, it remains a challenge to consistently obtain high-quality, intact, and flat cryostat sections onto glass slides because of the tiny sample size of the DRG tissue. So far, there is no article describing an optimal protocol for DRG cryosectioning. This protocol presents a step-by-step method to resolve the frequently encountered difficulties associated with DRG cryosectioning. The presented article explains how to remove the surrounding liquid from the DRG tissue samples, place the DRG sections on the slide facing the same orientation, and flatten the sections on the glass slide without curving up. Although this protocol has been developed for cryosectioning the DRG samples, it can be applied for the cryosectioning of many other tissues with a small sample size.

作者聚焦：小鼠背根神经节冷冻切片程序  

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

小鼠背根神经节冷冻切片的程序

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...

A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures

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A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures