isolation and culture of primary schwann cells from a human dermis

Isolation and Culture of Primary Schwann Cells from a Human Dermis

Precoat T25 flasks with poly-L-lysine or PLL, using 5 milliliters of DPBS, and place the precoated flasks at 37 degrees Celsius for three hours. Later, wash the PLL coating matrix twice with DPBS. After washing the isolated pieces of the dermis with 5 milliliters of DMEM basal medium, mince the dermis into small pieces with scissors.
Digest the minced dermis with 5 milliliters of collagenase at 37 degrees Celsius for 2 and 1/2 hours, with gentle trituration using a pipette tip every 30 minutes until the dermis dissociates completely. Once digested, filter the cell suspension with a 70-micrometer cell strainer, followed by dilution of the filtered cell suspension with 5 milliliters of complete DMEM medium to stop the enzymatic activity of collagenase.
Next, centrifuge the cell suspension at 870 times g for 5 minutes at room temperature, and resuspend the cell pellet in 2 milliliters of DMEM complete medium to divide the cells. After repeating the centrifugation process as described, aspirate the supernatant to use the cell pellet to isolate the Schwann cells or fibroblasts.
To isolate the Schwann cells, resuspend the cell pellet in 5 milliliters of fresh DMEM complete medium, and quantify the cell number and viability before seeding 5 milliliters of the resuspended cells in precoated T25 flasks at a density of 4.0 times 10 to the 3 cells per milliliter. Incubate the flask at 37 degrees Celsius and 5% carbon dioxide. After 16 hours, confirm cell adhesion by microscopy at 10x magnification. Then, remove the non-adherent cells, and wash the flask thrice with 5 milliliters of DPBS.
Next, incubate the cells with 5 milliliters of 10 micromolar cytosine arabinoside in a DMEM complete medium. After 24 hours, aspirate the cytosine arabinoside-containing medium before rinsing the flask. Refill the culture flask with 5 milliliters of Schwann cells complete culture medium to incubate for 48 hours while refreshing the medium every other day until Schwann cells reach 80% confluency.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Schwann cells (SCs) and fibroblasts from the dermis
<ol>
	<li>Pre-coat T25 flasks with poly-L-lysine (PLL, 0.01 &#181;g/&#181;L) using 5 mL of 1x DPBS. Place the pre-coated flasks at 37 &#176;C for 3 h. Wash the PLL coating matrix twice using 1x DPBS (5 mL). 
	NOTE: T25 flasks can be prepared ahead of time.</li>
	<li>Rinse the isolated pieces of the dermis with 5 mL of DMEM basal medium.</li>
	<li>Mince the dermis into small pieces with scissors or a scalpel.</li>
	<li>Digest the minced dermis with 5 mL of collagenase (2 mg/mL in DMEM basal medium) at 37 &#176;C for 2.5 h. Gently triturate the minced dermis with a pipette tip every 30 min until the dermis completely dissociates.</li>
	<li>Filter the cell suspension with a 70 &#181;M cell strainer. It is necessary to apply mechanical force using a 1 mL syringe to fully filter the suspension.</li>
	<li>Dilute the filtered cell suspension with 5 mL of complete DMEM medium (DMEM basal medium + 5% FBS + 1x of antibiotic) to stop the enzymatic activity of collagenase.</li>
	<li>Centrifuge the cell suspension at 870 x g for 5 min at RT to pellet the cells.</li>
	<li>From this cell pellet, both fibroblasts and SCs will be obtained. Resuspend the cell pellet in 2 mL of DMEM complete medium to divide the cells (1 mL cell suspension/tube) and centrifuge at 870 x g for 5 min at RT.</li>
	<li>Aspirate the supernatant. Resuspend the cell pellet in 5 mL of fresh DMEM complete medium.</li>
	<li>Quantify the cell number and viability. 
	NOTE: Here, a specialized cell sampling and staining device (Table of Materials) was used to quantify the cell number and viability following the manufacturer&#39;s instructions.</li>
	<li>Seed the pre-coated T25 flasks with 5 mL of resuspended cells at a density 4 x 103 cells/mL. Incubate the flask at 37 &#176;C in the presence of 5% CO2 overnight (~12-16 h incubation).</li>
	<li>After 16 h, confirm cell adhesion by microscopy (Table of Materials) at 10x magnification.</li>
	<li>Remove the non-adherent cells and wash the flask 3x with 5 mL of 1x DPBS.</li>
	<li>Add 5 mL of 10 &#181;M cytosine arabinoside (antimitotic agent, kills fibroblasts) in DMEM complete medium. Incubate the flask at 37 &#176;C in the presence of 5% CO2 for 24 h.</li>
	<li>Aspirate the cytosine arabinoside-containing medium from the flask. Rinse the flask 3x with 5 mL of 1x DPBS.</li>
	<li>Refill the culture flask with 5 mL of SCs complete culture medium (Table of Materials) and incubate at 37 &#176;C in the presence of 5% CO2 for 48 h. Refresh SC complete culture medium every other day until SCs reach 80% confluence.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;M sterile filters (Millex-GP Syringe Filter Unit,polyethersulfone)</td>
			<td>MilliporeSigma</td>
			<td>SLGPR33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;M cell strainers</td>
			<td>CELLTREAT</td>
			<td>229483</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;M cell strainers</td>
			<td>CELLTREAT</td>
			<td>229485</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL disposable syringes</td>
			<td>BD Luer-Lok</td>
			<td>BD-309659</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL disposable syringes (Syringe sterile, single use)</td>
			<td>BD Luer-Lok</td>
			<td>BD309646</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL disposable syringes</td>
			<td>BD Luer-Lok</td>
			<td>BD305462</td>
			<td></td>
		</tr>
		<tr>
			<td>1% TritonX-100</td>
			<td>Sigma</td>
			<td>X100-1L</td>
			<td>Prepared at the time of use.</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution</td>
			<td>ThermoFisher Scientific</td>
			<td>J19943.K2</td>
			<td>Ready to use and store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>5% BSA</td>
			<td>Sigma</td>
			<td>A7906-100G</td>
			<td>Prepared at the time of use.</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic</td>
			<td>ScienCell Research</td>
			<td>503</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemometec Vial1-Cassette</td>
			<td>Fisher Scientific</td>
			<td>NC1420193</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Gibco</td>
			<td>17018-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Fisherbrand</td>
			<td>12544D 22*50-1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase I</td>
			<td>Sigma-Aldrich</td>
			<td>D46693</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basal medium</td>
			<td>ScienCell Research</td>
			<td>9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline free from calcium and magnesium ions (DPBS)</td>
			<td>Corning</td>
			<td>21-031-CV)</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro-centrifuge tubes</td>
			<td>Fisherbrand</td>
			<td>02-681-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Buffered Saline Solution (HBSS buffer)</td>
			<td>Lonza</td>
			<td>CC-5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysisne (PLL)</td>
			<td>ScienCell Research</td>
			<td>32503</td>
			<td></td>
		</tr>
		<tr>
			<td>Schwann cell culture medium</td>
			<td>ScienCell Research</td>
			<td>1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision tweezers DUMONT straight with extra fine tips Dumostar, 5</td>
			<td>ROTH</td>
			<td>LH75.1</td>
			<td>Sterilize with 70% alcohol before use.</td>
		</tr>
		<tr>
			<td>IRIS Scissors, sharp/sharp. Length 4&#8211;3/8&#34;(111mm)</td>
			<td>Codman</td>
			<td>54-6500</td>
			<td>Sterilize with 70% alcohol before use.</td>
		</tr>
		<tr>
			<td>Sterilized surgical - sharp blade (Duro Edge Economy Single Edge Blades)</td>
			<td>Razor blade company</td>
			<td>94-0120</td>
			<td>Sterilize with 70% alcohol before use.</td>
		</tr>
		<tr>
			<td>T25 culture flask</td>
			<td>Corning</td>
			<td>353109</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin neutralization buffer (TNS)</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>ZEISS</td>
			<td>Primovert</td>
			<td>For visualizing/observing cell attachment or detachment.</td>
		</tr>
	</tbody>
</table>

Source: Jagadeeshaprasad, M. G., et al. <a href="https://app.jove.com/v/63776">Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin.</a> J. Vis. Exp. (2022)
This video demonstrates a protocol for isolating and culturing Schwann cells from human dermis tissues. The human dermis contains various cell types, including fibroblasts and Schwann cells. After isolation, the cells are treated with an antimitotic agent to eliminate proliferating fibroblasts, resulting in a pure culture of Schwann cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Schwann cells (SCs) ...

Take pieces of isolated human dermis containing various cell types, including fibroblasts, Schwann cells, and immune cells, embedded within a collagen matrix.
Mince the tissue into small fragments.
Add collagenase, a proteolytic enzyme, to digest the tissue&#39;s collagen fibers, loosening the cells.
Mechanically dissociate the tissue, obtaining a cell suspension.
Pass the cells through a cell strainer, removing the cell aggregates and tissue debris.
Add media containing serum to the cell suspension to stop the collagenase activity.
Centrifuge and discard the supernatant containing collagenase.
Resuspend the cells in media and plate them in a poly-L-lysine-coated culture flask.
Incubate for the cells to adhere to the coated surface.
Remove the media containing non-adherent cells. Wash with buffer.
Incubate with an antimitotic agent to eliminate rapidly dividing cells, like fibroblasts.
Remove media. Wash with buffer to remove any remaining antimitotic agents.&#160;
Add Schwann cell-specific culture media and incubate, facilitating the growth of Schwann cells.

14 ビュー. ヒト真皮からの初代シュワン細胞の単離と培養

ビデオ: ヒト真皮からの初代シュワン細胞の単離と培養 - 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

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

エレクトロポレーションによる齧歯類坐骨神経内のシュワン細胞へのインビボ遺伝子導入で

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

神経科学

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.

体外における感覚神経と運動神経からの線維芽細胞およびシュワン細胞の精製

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

Isolation and Culture of Primary Schwann Cells from a Human Dermis

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Isolation and Culture of Primary Schwann Cells from a Human Dermis