isolation of schwann cells from mouse spinal roots

Isolation of Schwann Cells from Mouse Spinal Roots

Slice the nerves into 3- to 5-millimeter pieces with scissors. Add 1 milliliter of 0.25% trypsin, and transfer the nerve pieces to 5-milliliter centrifuge tubes. Incubate at 37 degrees Celsius for 18 to 20 minutes. Next, to stop the digestion, add 3 to 4 milliliters of DMEM containing 10% fetal bovine serum. Pipette the mixture up and down gently, about 10 times. Centrifuge at 800 times g for 5 minutes.
After centrifugation, discard the supernatant, and resuspend the precipitate in 2 to 3 milliliters of DMEM supplemented with 10% FBS. Filter the cell suspension through a 400-mesh filter, and then use the suspension to inoculate a 60-millimeter Petri dish. Incubate the Petri dishes for 4 to 5 days at 37 degrees Celsius in the presence of 5% carbon dioxide.
When the culture has reached 90% confluence, wash the cells once using 1X PBS. Then, to digest the Schwann cells, add 1 milliliter of 0.25% trypsin at 37 degrees Celsius per 60-millimeter dish. After 8 to 10 seconds at room temperature, stop the digestion by adding 3 milliliters of DMEM supplemented with 10% FBS. To detach the Schwann cells, gently blow on the cells with a pipette.
Verify by microscope that the cells are detached. Then, collect the medium, which contains the Schwann cells, and centrifuge at 800 x g for 5 minutes. Discard the supernatant, and resuspend the precipitate in 3 milliliters of medium. Use the suspension to inoculate uncoded 60-millimeter Petri dishes, and incubate the dishes at 37 degrees Celsius for 30 to 45 minutes. Transfer the medium, which will contain the Schwann cells to a poly-L-lysine-coated medium dish, and incubate the dish at 37 degrees Celsius for 2 days.

isolation-of-schwann-cells-from-mouse-spinal-roots

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Isolation and culture of motor and sensory nerve fibroblasts and SC (Schwann cells)
<ol>
	<li>Use seven-day-old Sprague-Dawley (SD) rats (n=4) from the Experimental Animal Center of Nantong University of China. Place the rats in a tank containing 5% isoflurane for 2-3 minutes, allow the animals to breathe slowly and have no independent activity, and then sanitize using 75% ethanol before decapitation.</li>
	<li>Use scissors to cut the back skin for about 3 cm and remove the spinal column. Open the vertebral canal carefully to expose the spinal cord.</li>
	<li>Maintain the spinal cord in a 60 mm Petri dish with 2-3 mL of ice-cold D-Hanks&#39; balanced salt solution (HBSS).</li>
	<li>Based on the anatomical structure, excise the ventral root (motor nerve) and then the dorsal root (sensory nerve) under a dissecting microscope. Next, place them in an ice-cold D-Hanks&#39; balanced salt solution (HBSS).</li>
	<li>After removing the HBSS, slice the nerves into 3-5 mm pieces with scissors and digest with 1 mL of 0.25% (w/v) trypsin at 37 &#176;C for 18-20 min. Next, supplement with 3-4 mL of DMEM containing 10% fetal bovine serum (FBS) to stop the digestion.</li>
	<li>Pipette the mixture up and down gently about ten times and centrifuge at 800 x g for 5 min. After that, discard the supernatant and suspend the precipitate in 2-3 mL of DMEM supplemented with 10% FBS.</li>
	<li>Filter the cell suspension using a 400 mesh filter, then inoculate the cells in a 60 mm Petri dish and culture at 37 &#176;C in 5% CO2. After 4-5 days of culturing, isolate the passage 0 (p0) fibroblasts and SCs after reaching 90% confluence.</li>
	<li>Isolation and culture of SCs (Figure 1)
	<ol>
		<li>Wash the cells once using 1x PBS. Add 1 mL of 0.25% (w/v) trypsin (37 &#176;C) per 60 mm Petri dish to digest the cells for 8-10 s at room temperature. After that, 3 mL of DMEM supplemented with 10% FBS was added to stop the digestion.</li>
		<li>Gently blow the mixture with a pipette to detach the SCs. Then, collect and centrifuge the SCs at 800 x g for 5 min.</li>
		<li>Discard the supernatant and suspend the residue in 3 mL of DMEM with 10% FBS, 1% penicillin/streptomycin, 2 &#181;M forskolin, and 10 ng/mL HRG, and then inoculate the cells in an uncoated 60 mm Petri dish. After culturing at 37 &#176;C for 30-45 min, the fibroblasts (a few fibroblasts are digested with SCs) attach to the bottom of the dish.</li>
		<li>Transfer the supernatant (including the SCs) to another poly-L-lysine (PLL)--coated medium dish and culture at 37 &#176;C for 2 days.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>D-Hank&#39;s balanced salt solution</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus</td>
			<td>SZ2-ILST</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10099-141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% (w/v) trypsin</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dishes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: He, Q., et al., &#38; Ding, F. <a href="https://app.jove.com/v/60952">Purification of fibroblasts and schwann cells from sensory and motor nerves in vitro.</a> J. Vis. Exp. (2020)
This video outlines the isolation and culturing of Schwann cells from mouse spinal roots. Isolated cells are then cultured in specific media. Schwann cells are isolated via differential digestion and adherence. They are subsequently transferred to extracellular matrix-coated coverslips for attachment and proliferation.

<img alt="Figure 1" src="/files/ftp_upload/22594/22594_figure_1.jpg" /> 
Figure 1: The schematic diagram showing the separation and purification steps of sensory and motor Fibroblasts and SCs.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take a nerve root and chop it into small pieces.
Transfer to a digestive enzyme-containing tube and incubate.
The enzyme digests the extracellular matrix.
Add media containing serum to stop digestion.
Pipette the content repeatedly, then centrifuge and discard the supernatant.
Resuspend the pellet.
Pass the suspension through a strainer to remove undigested tissue. Incubate the collected cells in a plate.
Fibroblast and Schwann cells or SCs attach to the plate and divide.
Remove the unattached cells. Wash the attached cells with a phosphate buffer.
Add digestive enzymes to loosen the digestion-sensitive SCs. Stop digestion with media.
Completely detach SCs by adding media.
Transfer the cells to a tube and centrifuge. Discard the supernatant and resuspend the cells in SC-specific media.
Transfer the cells to an uncoated plate and incubate.
Any remaining fibroblasts will adhere, leaving SCs in suspension.
Transfer the suspension to a matrix-coated plate.
Incubate for Schwann cell attachment and division.

39 ビュー. マウス脊髄根からのシュワン細胞の単離

ビデオ: マウス脊髄根からのシュワン細胞の単離 - 実験

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

成熟シュワン細胞の発生源としての骨髄由来前駆細胞の低酸素プレコンディショニング

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

JoVE Journal

発生生物学

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

神経科学

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.

ラット背根神経節外植片とシュワン細胞の共培養における末梢軸索のin vitro髄鞘形成

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

Isolation of Schwann Cells from Mouse Spinal Roots

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Isolation of Schwann Cells from Mouse Spinal Roots