magnetic separation of schwann cells from mouse sciatic nerves

Magnetic Separation of Schwann Cells from Mouse Sciatic Nerves

Transfer the digested nerves into a 50-milliliter tube to centrifuge at 188 g for 10 minutes at 4 degrees Celsius. After discarding the supernatant, resuspend the pellet in 10 milliliters of DMEM containing 10% FCS and 50 micrograms per milliliter of gentamicin.
Next, filter the cell suspension through a 100-micrometer cell strainer. After centrifuging at 188 g for 10 minutes at 4 degrees Celsius, resuspend the pellet with 4 milliliters of DMEM containing FCS and gentamicin. Add 2 milliliters of the cell suspension to each of the two poly-L-lysine and laminin-coated 60-millimeter tissue culture dishes. Incubate at 37 degrees Celsius and 5% carbon dioxide, leaving the plates untouched for 2 days in the incubator.
Centrifuge the Schwann cell suspension at 188 g for 10 minutes at 4 degrees Celsius, and resuspend the cell pellet in 90 microliters of magnetic cell separation buffer per 1 x 107 cells. Then, add 10 microliters of Thy-1 microbeads per 1 x 107 cells. Incubate the resuspended solution for 15 minutes in the dark at 8 degrees Celsius.
Next, add 2 milliliters of the magnetic cell separation buffer to the cell suspension. After centrifuging at 300 g for 10 minutes at 4 degrees Celsius, resuspend the pellet in 500 microliters of magnetic cell separation buffer. Place the magnetic cell separation column in the cell separator, and moisten the column with 1 milliliter of the magnetic cell separation buffer. Then, apply the cells to it to collect the flow through.

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1. Schwann cell culture
<ol>
	<li>Coating for Schwann cell culture
	<ol>
		<li>Coat the cell culture dishes under sterile conditions. Apply 2 mL of 0.01% poly-L-lysine (PLL) to two 60 mm tissue culture (TC) dishes each and incubate overnight at 4 &#176;C.</li>
		<li>Remove the PLL, wash the TC dishes 2x with distilled water, and incubate with 2 mL of 1 &#181;g/cm2 laminin overnight at 4 &#176;C. Wash the TC dishes 2x with aqua dest, and let the plates air-dry.</li>
	</ol>
	</li>
	<li>Medium preparation for Schwann cell culture
	<ol>
		<li>Prepare 50 mL of Schwann cell medium by adding 10% heat-inactivated fetal calf serum (FCS), 2 &#181;M forskolin, 10 nM neuregulin, and 50 &#181;g/mL gentamycin to Dulbecco&#8242;s Modified Eagle&#8242;s Medium (DMEM)/F-12 (high glucose) under sterile conditions.</li>
		<li>Prepare 70 mL of Leibovitz&#39;s L-15 medium with 50 &#181;g/mL gentamycin under sterile conditions.</li>
	</ol>
	</li>
	<li>Enzymatic digestion of the sciatic nerve 
	&#8203;NOTE: The next steps are performed under sterile conditions.
	<ol>
		<li>Prepare the enzymatic digestion solution containing 0.25% dispase II, 0.05% type I collagenase, and 50 &#181;g/mL gentamycin in 10 mL of DMEM (high glucose).</li>
		<li>Centrifuge the tube at 188 x g for 5 min at 4 &#176;C, remove the supernatant with a 25 mL serological pipette, and transfer the pellet with the remaining Leibovitz&#39;s L-15 medium into a 60 mm tissue culture dish using a 1,000 mL pipette.</li>
		<li>Rinse the 50 mL tube with 10 mL of the enzymatic digestion solution and add it to the dish containing the nerve fibers. Distribute the tissue in the dish carefully with the tip of a pipette to maximize the accessible surface for digestion.</li>
		<li>Incubate at 37 &#176;C and 5% CO2 for 18 h, and stop the digestion by adding 10 mL of 40% FCS in Hanks&#39; balanced salt solution, without Ca2+ and Mg2+ (HBSS).</li>
	</ol>
	</li>
	<li>Cell separation
	<ol>
		<li>Transfer the digested nerves into a 50 mL tube using a serological pipette and centrifuge at 188 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 10 mL of DMEM containing 10% FCS and 50 &#181;g/mL gentamycin. Resuspend the pellet 20 times subsequently, using a 10 mL, 5 mL, 2 mL, 1 mL, and 200 &#181;L pipette tip.</li>
		<li>Filter the cell suspension through a 100 &#181;m cell strainer and centrifuge at 188 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the pellet with 4 mL of DMEM containing 10% FCS and 50 &#181;g/mL gentamycin.</li>
		<li>Add 2 mL of the cell suspension to each of the two PLL- and laminin-coated 60 mm TC dishes, and incubate at 37 &#176;C and 5% CO2. Leave the plates untouched for 2 days in the incubator to protect the cells from mechanical stress and to support adherence.</li>
	</ol>
	</li>
	<li>Schwann cell differentiation
	<ol>
		<li>After 2 days, remove the medium and carefully rinse the plates 2x with DMEM (high glucose), 10% FCS, and 50 &#181;g/mL gentamycin. Afterward, add 2 mL of Schwann cell medium. Replace the Schwann cell medium every 2nd day, and observe the cell appearance and confluency using a microscope.</li>
	</ol>
	</li>
	<li>Cell trypsinization and magnetic separation
	<ol>
		<li>When the cells reach a confluency of about 80% (6-12 days of culture), carefully wash the plates 2x with 3 mL of DPBS and incubate with 2 mL of 0.05% Trypsin/EDTA (prewarmed to 37 &#176;C) for 3 min. When the cells detach from the plate bottom, inactivate digestion by the addition of 2 mL of DMEM with 10% FCS and 50 &#181;g/mL gentamycin. 
		NOTE: Stick to a trypsinization time of strictly 3 min, and proceed rapidly afterward.</li>
		<li>Resuspend the cell pellet in 2 mL of magnetic cell separation buffer containing DPBS with 0.5% bovine serum albumin (BSA) and 2 nM EDTA. Combine 10 &#181;L of the cell suspension with 10 &#181;L of trypan blue and count the cells using a staining chamber.</li>
		<li>Centrifuge the cell suspension at 188 x g for 10 min at 4 &#176;C and resuspend the cell pellet in 90 &#181;L of magnetic cell separation buffer per 1 x 107 cells. Add 10 &#181;L of Thy-1 microbeads per 1 x 107 cells. Resuspend the solution a few times and incubate for 15 min in the dark at 8 &#176;C.</li>
		<li>Add 2 mL of the magnetic cell separation buffer to the cell suspension and centrifuge at 300 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 500 &#181;L of magnetic cell separation buffer.</li>
		<li>Moisten the magnetic cell separation column with 1 mL of the magnetic cell separation buffer. Place the magnetic cell separation column in the magnetic cell separator. Apply the cells to the magnetic cell separation column. Collect the flow through and centrifuge at 300 x g at 4 &#176;C for 10 min. 
		&#8203;NOTE: Fibroblasts are positively selected and remain in the column, while Schwann cells pass the column. Fibroblasts can be collected with a stamp (e.g., as a negative control for Schwann cell staining protocols).</li>
		<li>Discard the supernatant and resuspend the pellet in 1 mL of the coculture medium. Count the cells after staining with trypan blue and a staining chamber.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>8076.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 100 &#181;M</td>
			<td>BD Bioscience, Heidelberg, Germany</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810-R</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>5811000015</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Heracell</td>
			<td>Heraeus Instruments, Hanau, Germany</td>
			<td>51017865</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water (Water Purification System)</td>
			<td>Millipore, Molsheim, France</td>
			<td>ZLXS5010Y</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany</td>
			<td>31331093</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (no calcium ions and no magnesium ions)</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany</td>
			<td>D8537-6X500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany</td>
			<td>F7524</td>
			<td>FCS must be tested for Schwann cell culture</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany</td>
			<td>5710064</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (no calcium ions and no magnesium ions)</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany</td>
			<td>14170138</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Multistand</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130042303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Motic, Wetzlar, Germany</td>
			<td>Motic BA 400</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>VWR, Radnor, USA</td>
			<td>630-1985</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130091632</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Assistant, Erlangen, Germany</td>
			<td>40441</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>2231300004</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany</td>
			<td>P4707-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>861254025</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>861685001</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 5 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>861253001</td>
			<td></td>
		</tr>
		<tr>
			<td>TC dish 60, cell +</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>833901300</td>
			<td></td>
		</tr>
		<tr>
			<td>Thy-1 Microbeads (MACS Kit)</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-094-523</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Type I Collagenase</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany</td>
			<td>C1639</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath type 1008</td>
			<td>GFL, Burgwedel, Germany</td>
			<td>4285</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Blusch, A., et al., <a href="https://app.jove.com/v/64768">In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells.</a> J. Vis. Exp. (2023)
This video demonstrates a method for isolating Schwann cells from mouse sciatic nerve fibers.

1. Schwann cell culture

	Coating for Schwann cell culture
	
		Coat the cell culture dishes under sterile conditions. Apply 2 mL of 0.01% poly-L-lysine ...

Take a suspension of cells and debris from enzymatically digested mouse sciatic nerve fibers.
Pass through a strainer, removing debris.
Centrifuge the filtrate and remove the supernatant.
Resuspend the cells in serum-containing media and seed them onto an adhesion protein-coated culture plate.
Incubate for cell adhesion. Replace media with Schwann cell media. Incubate for Schwann cell monolayer formation.
Remove the media and wash with buffer. Incubate with trypsin to detach cells.
Add media containing serum to stop the enzymatic activity.
Collect the cells and centrifuge. Discard the supernatant.
Resuspend the cells in buffer and add antibody-coated magnetic microbeads.
Incubate to bind antibodies to fibroblast glycoproteins.
Centrifuge and discard the supernatant. Resuspend the cells in buffer, and load them into a magnetic separation column containing ferromagnetic spheres placed in the magnetic field of the magnetic separator.
The magnetic field retains the microbead-bound fibroblasts, allowing Schwann cells to be collected in the flow-through.

22 조회수. 마우스 좌골 신경에서 슈반 세포의 자기 분리

비디오: 마우스 좌골 신경에서 슈반 세포의 자기 분리 - 실험

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.

성숙한 슈반 세포 생성을위한 골수 유래 선조 세포의 저산소 전처리

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

연구

JoVE Journal

발생학

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

체외에서 감각 및 모터 신경에서 섬유 아세포와 슈완 세포의 정화

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

신경과학

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.

인간 포피로부터의 일차 슈완 세포, 각질세포 및 섬유아세포의 분리, 배양 및 특성화

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

Magnetic Separation of Schwann Cells from Mouse Sciatic Nerves

내레이션 대본

Magnetic Separation of Schwann Cells from Mouse Sciatic Nerves