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Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons
1. Preparation of purified rat DRG neurons
<ol>
	<li>Autoclave all dissection tools (i.e., dissection scissors, forceps, two microdissection forceps, and microdissecting scissors) at 180 &#176;C for at least 2 h prior to use.</li>
	<li>Coat 6-well tissue culture plates with poly-D-lysine (PDL, 10 &#181;g/mL in PBS) at 4&#176;C overnight. Remove the PDL and rinse with 1.5 m of PBS per well.</li>
	<li>Proceed with coating the plates with laminin (10 &#181;g/mL in PBS) at 37 &#176;C for 2 h. Rinse the plates with 1.5 mL of PBS per well.</li>
	<li>Prepare DRG neuron maintenance medium, comprised of neurobasal medium supplemented with B27 (2% v/v), L-glutamine (1% v/v), nerve growth factor (NGF, 20 ng/mL), and penicillin/streptomycin (P/S, 1% v/v).</li>
	<li>Prepare DRG neuron purification medium, comprised of neurobasal medium supplemented with B27 (2% v/v), L-glutamine (1%), nerve growth factor (NGF, 20 ng/mL), P/S (1%), fluorodeoxyuridine (FDU, 10 &#181;g/mL), and uridine (10 &#181;g/mL).</li>
	<li>Sacrifice pregnant rats at gestational day 14-15 by pentobarbital overdose (240 mg/kg bodyweight, intraperitoneal).</li>
	<li>Place the sacrificed animals in supine position. Clean their abdomen thoroughly with 70% ethanol.</li>
	<li>Cut the lower abdominal wall of the animal longitudinally using fine dissecting scissors and forceps. Identify and remove the uterus using dissection scissors. Cut the uterine wall to expose and extract the embryos. Transfer the embryos to a sterile, 10 cm culture dish filled with PBS. Place the culture dish on ice.</li>
	<li>Transfer the embryo intended for dissection to a sterile, 10 cm culture dish filled with PBS (room temperature) and position it beneath a dissection microscope. Have the embryo in prone position. 
	NOTE: The whitish spinal cord and the attached DRGs can be seen over the dorsal aspect of the embryo, through its translucent skin.</li>
	<li>Insert microdissecting forceps along either side of the spinal cord and use blunt dissection to begin separating the spinal cord from the surrounding soft tissue. Cut the spinal cord free from the animal using microdissecting forceps along the neck opening and tail stub. Perform further blunt dissection over the ventral aspect of the cord to free it from surrounding soft tissue.</li>
	<li>Use microdissecting forceps to remove residual soft tissue over the dorsal aspect of the freed spinal cord. 
	NOTE: At this stage, only the spinal cord, nerve roots, and attached DRG should remain.</li>
	<li>Detach individual DRGs from their connecting nerve roots using microdissecting forceps. Use a pipette pen attached to a 1 mL tip to transfer the DRGs to a 1.5 mL, sterile centrifuge tube containing PBS. 
	NOTE: For each 1.5 mL tube, a maximum of 100 DRGs can be accommodated.</li>
	<li>Centrifuge the DRGs at 250 x g for 5 min and resuspend them in recombinant enzymatic cell dissociation reagent (200 &#181;L per tube). Incubate (37 &#176;C, 5% CO2) for 10 min. Centrifuge the DRGs at 250 x g for 5 min, remove the supernatant, and resuspend in DRG neuron maintenance medium. Dissociate the pellet by gentle trituration using a 200 &#181;L pipette tip. Quantify the cells within the pellet using a hemocytometer after an appropriate dilution.</li>
	<li>Seed the cells at a density of 5,000 cells/cm2 into PDL/laminin-coated 6-well plates in 1.5 mL of DRG neuron maintenance medium per well. After two days of culture, remove the DRG neuron maintenance medium, rinse with PBS, and replace with DRG neuron purification medium. 
	NOTE: For each purification cycle, treat the DRG cultures with purification medium for 2 days, followed by 1 day of incubation in maintenance medium. After 3-4 purification cycles, the removal of all endogenous glia is expected. This should take approximately 14 days. Purified cultures test positive for the neuronal marker TUJ1 and are absent for S100&#946; expression (Figure 1).</li>
</ol>
2. Generation of Schwann cell-like cells
<ol>
	<li>Prepare glial induction medium comprised of &#945;MEM (Minimum essential medium Eagle (MEM), alpha modifications) supplemented with &#946;-Heregulin (100 ng/mL), basic fibroblast growth factor (bFGF, 10 ng/ml), platelet-derived growth factor (PDGF-AA, 5 ng/mL), fetal bovine serum (FBS, 10%), and P/S (1% v/v).</li>
	<li>Plate the neurospheres prepared in section 4 in PDL/laminin-coated 6-well plates at a density of 5-10 spheres per cm2 in 1.5 mL of glial induction medium per well. Replace glial induction medium every 2 days after rinsing the cells with PBS.</li>
	<li>NOTE: Cells from seeded neurospheres are seen to migrate outwards by day 2. By day 7, migratory cells have a tapered appearance and should demonstrate immunopositivity for the Schwann cell markers p75 neurotrophin receptor (p75) and S100&#946;7. These cells are referred to as SCLCs.</li>
</ol>
3. Coculture of SCLCs with DRG neurons
<ol>
	<li>Prepare coculture medium comprised of DRG neuron maintenance medium (step 1.1.4) and glial induction medium (step 1.2.1) at a 1:1 volume-to-volume ratio.</li>
	<li>Prepare Schwann cell maintenance medium comprised of Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)) supplemented with FBS (5%), &#946;-Heregulin (10 ng/mL), and P/S (1% v/v).</li>
	<li>Remove the culture medium from day 7 SCLCs, rinse them with PBS, and incubate them with 0.5 mL/well of recombinant enzymatic cell dissociation reagent at 37 &#176;C for 5 min. Resuspend the SCLCs in coculture medium.</li>
	<li>Quantify the cells using a hemocytometer after an appropriate dilution.</li>
	<li>Seed the SCLCs onto purified DRG neuron cultures at a density of 1,000 cells/cm2. Maintain the cocultures for 14 days, with medium replacement every 2 days. 
	NOTE: During coculture, SCLCs acquired the spindle-like morphology that is typical of mature Schwann cells (Figure 2). These cells persist in their phenotype after the withdrawal of growth factors and are able to myelinate axons in vitro and in vivo. The positivity of Schwann cell markers (i.e., p75 and S100&#946;) should be monitored by immunofluorescence.</li>
	<li>Upon completion of coculture, passage fate-committed Schwann cells. Use 0.5 mL of dissociation reagent per well. Quantify the cells using a hemocytometer after an appropriate dilution.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#945;MEM</td>
			<td>Sigmaaldrich</td>
			<td>M4526</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher scientific</td>
			<td>12400-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Thermofisher scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biosera</td>
			<td>FB-1280/500</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher scientific</td>
			<td>17504-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Thermofisher scientific</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor (bFGF)</td>
			<td>Peprotech</td>
			<td>100-18B/100UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve growth factor (NGF)</td>
			<td>Millipore</td>
			<td>NC011</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet-derived growth factor-AA (PDGF-AA)</td>
			<td>Peprotech</td>
			<td>100-13A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heregulin beta-3, EGF domain (&#946; Her)</td>
			<td>Millipore</td>
			<td>01-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigmaaldrich</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Fluro-2&#39; - deoxyuridine (FDU)</td>
			<td>Sigmaaldrich</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine (PDL)</td>
			<td>Sigmaaldrich</td>
			<td>P7886-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermofisher scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermofisher scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin (P/S)</td>
			<td>Thermofisher scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Thermofisher scientific</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm plate for adherent culture</td>
			<td>TPP</td>
			<td>93100</td>
			<td>Used for selection of MSCs by tissue culture adherence</td>
		</tr>
		<tr>
			<td>6-well plate for adherent culture</td>
			<td>TPP</td>
			<td>92006</td>
			<td>Used for expansion of MSCs following passaging</td>
		</tr>
		<tr>
			<td>UltraLow 6-well plate for non adherent culture</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Used for neural progenitor enrichment</td>
		</tr>
		<tr>
			<td>Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td>MIC-101</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigmaaldrich</td>
			<td>H4034-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of drg neurons and coculture with schwann cell precursors to generate schwann cells

Source: Tsui, Y, et al. <a href="https://app.jove.com/v/55794">Hypoxic Preconditioning of Marrow-derived Progenitor Cells as a Source for the Generation of Mature Schwann Cells.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure for isolating dorsal root ganglion (DRG) neurons from rat embryos and co-culturing them with Schwann cell precursors to generate mature Schwann cells. The isolated DRGs are processed to isolate neurons and glial cells. The isolated cells are cultured to purify the neurons, which are then co-cultured with Schwann cell-like cells (SCLCs) in a co-culture medium. Interaction with the DRG neurons leads to the maturation of SCLCs into Schwann cells.

<img alt="Figure 1" src="/files/ftp_upload/22394/22394_Figure1.jpg" /> 
Figure 1: Establishing purified rat DRG networks. Purified DRG networks are established after pulsed treatment with the antimitotic agents FDU and uridine (A). Neurite networks that are devoid of S100&#946;-expressing endogenous glia (B) are ready for coculture with SCLCs. Scale bars = 100 &#181;m.
<img alt="Figure 2" src="/files/ftp_upload/22394/22394_Figure2.jpg" /> 
Figure 2: Generation of bone marrow-derived Schwann cells via coculture with DRG neurons.&#160;Upon completing 2 weeks of coculture with purified DRG neurons and human SCLCs, spindle-shaped, fate-committed Schwann cells emerge from both normoxia- and hypoxia-treated groups (A&#160;and&#160;D). These cells express the Schwann cell markers p75 (B and E) and S100&#946; (C and F). The expression of human nuclei antigen (HuNeu) demonstrates that the S100&#946;-positive cells were not contaminating glial cells originating from rat DRGs. Scale bars = 100 &#181;m.

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

Generation of Fate-committed Schwann Cells via Coculture with DRG Neurons
1. Preparation of purified rat DRG neurons

	Autoclave all dissection tools ...

Take the spinal cord of a rat embryo with attached dorsal root ganglions, or DRGs, containing neurons and glial cells.
Detach individual DRGs from the cord, transfer them to a tube, and centrifuge, discarding any tissue debris.
Add enzymes to dissociate DRG neurons and glial cells.
Spin down the cells and remove any debris. Add a neuronal maintenance medium and triturate to generate a single-cell suspension.
Transfer the suspension into a microplate coated with a culture substrate, promoting cell adherence.
Introduce a purification medium and incubate. The medium&#39;s inhibitory agents eliminate dividing glial cells while non-dividing neurons survive.
Take Schwann cell-like cells, or SCLCs, in a co-culture medium. SCLCs, the precursor of Schwann cells, interact with embryonic peripheral nerve neurons.
Introduce the suspension onto the DRG neurons and incubate. Interaction of the SCLCs with DRG neurons promotes their maturation into Schwann cells.
The generated Schwann cells are ready for analysis.

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Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

154 Views. Source: Tsui, Y, et al. Hypoxic Preconditioning of Marrow-derived Progenitor Cells as a Source for the Generation of Mature Schwann Cells. J. Vis. Exp. (2017). This video demonstrates a procedure for isolating dorsal root ganglion (DRG) neurons from rat embryos and co-culturing them with Schwann cell precursors to generate mature Schwann cells. The isolated DRGs are processed to isolate neurons and glial cells. The isolated cells are cultured to purify the neurons, which are then co-cultured wi...

Video: Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells - Concept

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.

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

JoVE Journal

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.

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

Mouse Dorsal Root Ganglion Cryosectioning

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.

Author Spotlight: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning  

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

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

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

Transcript

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

Rapid Isolation of Dorsal Root Ganglion Macrophages

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

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning