This study was supported by the National Key Research and Development Program of China (Grant No. 2017YFA0104703), the National Natural Foundation of China (Grant No. 31500927).

This study was carried out in accordance with the Institutional Animal Care Guidelines of Nantong University. All the procedures including the animal subjects were ethically approved by the Administration Committee of Experimental Animals, Jiangsu Province, China.
1. Isolation and culture of motor and sensory nerve fibroblasts and SCs
<ol>
	<li>Use seven-day-old Sprague-Dawley (SD) rats (n=4) provided by 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 breath slowly and have no independent activity, and then sanitize using 75% ethanol prior to 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 10 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, and then inoculate the cells in 60 mm Petri dish and culture at 37 &#176;C in the presence of 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, add 3 mL of DMEM supplemented with 10% FBS to stop the digestion.</li>
		<li>Gently blow the mixture to detach the SCs with a pipette. Then collect and centrifuge the SCs at 800 x g for 5 min.</li>
		<li>Discard the supernatant and suspend the precipitate 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 uncoated 60 mm Petri dish. After culturing at 37 &#176;C for 30-45 min, the fibroblasts (a few number of 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>
	<li>Isolation and culture of fibroblasts (Figure 1)
	<ol>
		<li>After removing the SCs (as shown in step 1.8), wash the remaining fibroblasts in the dishes with 1x PBS and then add 1 mL of 0.25% (w/v) trypsin to digest the fibroblasts for 2 min at 37 &#176;C.</li>
		<li>Add DMEM supplemented with 10% FBS to end the digestion. Blow the fibroblasts using a pipette, and then collect and centrifuge them at 800 x g for 5 min.</li>
		<li>Discard the supernatant, suspend the precipitate with 2 mL of DMEM containing 10% FBS, and then inoculate the cells in uncoated 60 mm Petri dish. The fibroblasts after culturing for 30-45 min at 37 &#176;C attach to the bottom of the dish. Discard the supernatant (including a few numbers of SCs). Then add 3 mL of DMEM supplemented with 10% FBS into the fibroblasts dish and culture at 37 &#176;C for 2 days.</li>
	</ol>
	</li>
	<li>Passage the p1 cells until they reached 90% confluence. Then purify them by differential digestion and differential adherence again as described in sections 1.8 and 1.9.</li>
	<li>Digest the p2 fibroblasts and SCs after culturing for 2 days, and then collect the cells, count and inoculate in 1 x 105 numbers/well on PLL-coated slides for immunocytochemistry (ICC).</li>
</ol>
2. ICC for identification of cell purity
<ol>
	<li>Culture the cells for 24 h at 37 &#176;C and perform ICC staining after differential digestion and differential adherence of motor and sensory fibroblasts and SCs.</li>
	<li>Wash the motor and sensory fibroblasts and SCs with 1x PBS and fix them in 200 &#181;L/well of 4% paraformaldehyde (pH 7.4) for 18 min at room temperature.</li>
	<li>Block the sample SCs with blocking buffer (0.1% Triton X-100 in 0.01 M PBS containing 5% goat serum) and block the sample fibroblasts with blocking buffer (0.01 M PBS containing 5% goat serum) for 45 min at 37 &#176;C after washing them with PBS thrice.</li>
	<li>Remove the blocking buffer, and incubate with the following primary antibodies: mouse monoclonal anti-CD90 antibody (a specific marker for fibroblasts) (1:1000) for fibroblasts and mouse anti-S100 antibody (a specific marker for SCs) (1:400) for SCs at 4 &#176;C overnight.</li>
	<li>Discard the primary antibodies, wash with PBS thrice, and incubate with the following secondary antibodies: Alexa Fluor 594 goat anti-mouse IgG (1:400) for fibroblasts and 488-conjugated goat anti-mouse IgG (1:400) for SCs at room temperature for 1.5 h.</li>
	<li>Wash the samples thrice with PBS, and stain the nuclei with 5 &#181;g/mL Hoechst 33342 dye for 10 min at room temperature. Wash the sample with 1x PBS thrice and mount them using the mounting medium (20 &#181;L/slide) on the glass slide.</li>
	<li>Take the cell photographs by confocal laser scanning microscope in three random fields for each well. Evaluate the total number of nuclei and CD90-positive cells, S100-positive cells and then calculate the percentage of CD90-positive cells and S100-positive cells, respectively. Perform the staining in triplicate.</li>
</ol>
3. Flow cytometry analysis (FCA) for identification of cell purity
<ol>
	<li>Evaluate the purity of motor and sensory fibroblasts and SCs as described previously by FCA16. Briefly, digest the p2 motor and sensory fibroblasts and SCs with 0.25% (w/v) trypsin, resuspend the cell pellets and incubate them in fixation medium at room temperature for 15 min.</li>
	<li>Incubate the cells with permeabilization medium and probe using mouse monoclonal anti-CD90 antibody (0.1 &#181;g/106 cells, 200 &#181;L) for fibroblasts and mouse anti-S100 antibody (1:400, 200 &#181;L) for SCs at room temperature for 30 min, respectively.</li>
	<li>Incubate the cells with 488-conjugated goat anti-mouse IgG for 30 min. Use FACS caliber to perform flow cytometry, and analyze the data using Cell Quest software.</li>
	<li>Incubate the cells only with 488-conjugated goat anti-mouse IgG (fibroblasts Group) and mouse IgG1 kappa [MOPC-21] (FITC) - Isotype control (SCs group), which serves as negative control.</li>
</ol>
4. Statistical analysis
<ol>
	<li>Present all data as means &#177; SEM. Assess statistical differences in the data by unpaired t-test using GraphPad Prism 6.0. Set statistical significance at p&#60;0.05. Perform all assays in triplicate.</li>
</ol>

<ol>
	<li>Stierli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+the+homeostasis+and+regeneration+of+peripheral+nerve+is+distinct+from+the+CNS+and+independent+of+a+stem+cell+population.">The regulation of the homeostasis and regeneration of peripheral nerve is distinct from the CNS and independent of a stem cell population.</a> Development (The Company of Biologists). , (2018).</li><li>Schubert, T., Friede, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endoneurial+fibroblasts+in+myelin+degradation.">The role of endoneurial fibroblasts in myelin degradation.</a> Journal of Neuropathology and Experimental Neurology. 40 (2), 134-154 (1981).</li><li>Dreesmann, L., Mittnacht, U., Lietz, M., Schlosshauer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+fibroblast+impact+on+Schwann+cell+behavior.">Nerve fibroblast impact on Schwann cell behavior.</a> European Journal of Cell Biology. 88 (5), 285-300 (2009).</li><li>Lavdas, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+engineered+to+express+the+cell+adhesion+molecule+L1+accelerate+myelination+and+motor+recovery+after+spinal+cord+injury.">Schwann cells engineered to express the cell adhesion molecule L1 accelerate myelination and motor recovery after spinal cord injury.</a> Experimental Neurology. 221 (1), 206-216 (2010).</li><li>Parrinello, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphB+signaling+directs+peripheral+nerve+regeneration+through+Sox2-dependent+Schwann+cell+sorting.">EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting.</a> Cell. 143 (1), 145-155 (2010).</li><li>Benito, C., Davis, C. M., Gomez-Sanchez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+Controls+the+Long-Term+Survival+and+Phenotype+of+Repair+Schwann+Cells+during+Nerve+Regeneration.">STAT3 Controls the Long-Term Survival and Phenotype of Repair Schwann Cells during Nerve Regeneration.</a> Journal of Neuroscience Research. 37 (16), 4255-4269 (2017).</li><li>Zhang, Z. J., Jiang, B. C., Gao, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+neuron-glial+cell+interaction+and+pathogenesis+of+neuropathic+pain.">Chemokines in neuron-glial cell interaction and pathogenesis of neuropathic pain.</a> Cellular and Molecular Life Sciences. 74 (18), 3275-3291 (2017).</li><li>Hoke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+express+motor+and+sensory+phenotypes+that+regulate+axon+regeneration.">Schwann cells express motor and sensory phenotypes that regulate axon regeneration.</a> Journal of Neuroscience. 26 (38), 9646-9655 (2006).</li><li>Brushart, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+phenotype+is+regulated+by+axon+modality+and+central-peripheral+location,+and+persists+in+vitro.">Schwann cell phenotype is regulated by axon modality and central-peripheral location, and persists in vitro.</a> Experiment Neurology. 247, 272-281 (2013).</li><li>He, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Gene+Expression+in+Primary+Cultured+Sensory+and+Motor+Nerve+Fibroblasts.">Differential Gene Expression in Primary Cultured Sensory and Motor Nerve Fibroblasts.</a> Frontiers in Neuroscience. 12, 1016 (2018).</li><li>Weinstein, D. E., Wu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Unit+17:+Isolation+and+purification+of+primary+Schwann+cells.">Chapter 3, Unit 17: Isolation and purification of primary Schwann cells.</a> Current Protocols in Neuroscience. , (2001).</li><li>Palomo Irigoyen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Purification+of+Primary+Rodent+Schwann+Cells.">Isolation and Purification of Primary Rodent Schwann Cells.</a> Methods in Molecular Biology. 1791, 81-93 (2018).</li><li>Cheng, L., Khan, M., Mudge, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcitonin+gene-related+peptide+promotes+Schwann+cell+proliferation.">Calcitonin gene-related peptide promotes Schwann cell proliferation.</a> Journal of Cell Biology. 129 (3), 789-796 (1995).</li><li>Pannunzio, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+selecting+Schwann+cells+from+adult+mouse+sciatic+nerve.">A new method of selecting Schwann cells from adult mouse sciatic nerve.</a> Journal of Neuroscience Methods. 149 (1), 74-81 (2005).</li><li>Shen, M., Tang, W., Cao, Z., Cao, X., Ding, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+Schwann+cells+based+on+cell+sorting.">Isolation of rat Schwann cells based on cell sorting.</a> Molecular Medicine Reports. 16 (2), 1747-1752 (2017).</li><li>He, Q., Man, L., Ji, Y., Ding, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+in+the+biological+characteristics+between+primary+cultured+sensory+and+motor+Schwann+cells.">Comparison in the biological characteristics between primary cultured sensory and motor Schwann cells.</a> Neuroscience Letters. 521 (1), 57-61 (2012).</li><li>Weiss, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+and+transcriptomics+of+peripheral+nerve+tissue+and+cells+unravel+new+aspects+of+the+human+Schwann+cell+repair+phenotype.">Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype.</a> Glia. 64 (12), 2133-2153 (2016).</li></ol>

The authors have nothing to disclose.

The two major cell populations of peripheral nerves included SCs and fibroblasts. The primarily cultured fibroblasts and SCs can accurately assist in modeling the physiology of fibroblasts and SCs during peripheral nerve development and regeneration. The study showed that P7 rat sciatic nerve cells contained about 85% of S100-positive SCs, 13% of OX7-positive fibroblasts and only 1.5% of OX42-positive macrophages13. Although the number of fibroblasts is less than SCs, the initial proliferation rat...

In the peripheral nervous system, the nerve fibers mainly consists of axons, Schwann cells (SCs), and fibroblasts, and also contains a small number of macrophages, microvascular endothelial cells, and immune cells1. SCs wrap the axons in a 1:1 ratio and are enclosed by a connective tissue layer called the endoneurium. The axons are then bundled together to form groups called fascicles, and each fascicle is wrapped in a connective tissue layer known as the perineurium. Finally, the whole nerve fiber is wrapped in a layer of connective tissue, which is termed as the epineurium. In the endoneurium, the whole cell population is comprised of 48% SCs, and a substantial portion of the remaining cells involves fibroblasts2. Furthermore, fibroblasts are important components of all nerve compartments, including the epineurium, the perineurium, and the endoneurium3. Many studies have indicated that SCs and fibroblasts play a crucial role in the regeneration process after peripheral nerve injuries4,5,6. After transection of the peripheral nerve, the perineurial fibroblasts regulate cell sorting via the ephrin-B/EphB2 signaling pathway between SCs and fibroblasts, further guiding the axonal regrowth through wounds5. Peripheral nerve fibroblasts secrete tenascin-C protein and enhance the migration of SCs during nerve regeneration through &#946;1-integrin signaling pathway7. However, the SCs and fibroblasts used in the above studies were derived from the sciatic nerve, which includes both sensory and motor nerves.
In the peripheral nervous system, the sensory nerves (afferent nerves) conduct sensory signaling from the receptors to the central nervous system (CNS), while the motor nerves (efferent nerves) conduct signals from the CNS to the muscles. Previous studies have indicated that SCs express distinct motor and sensory phenotypes and secrete neurotrophic factors to support peripheral nerve regeneration8,9. According to a recent study, fibroblasts also express different motor and sensory phenotypes and affect the migration of SCs10. Thus, the exploration of differences between motor and sensory nerve fibroblasts/SCs allows us to study the complicated underlying molecular mechanisms of peripheral nerve specific regeneration.
At present, there are many ways to purify SCs and fibroblasts, including the application of antimitotic agents, antibody-mediated cytolysis11,12, sequential immunopanning13 and laminin substratum14. However, all the above methods remove fibroblasts and preserve only the SCs. Highly purified SCs and fibroblasts can be obtained by flow cytometry sorting technology15, but it is a time-consuming and costly technique. Hence, in this study, a simple differential digestion and differential adherence method for purifying and isolating sensory and motor nerve fibroblasts and SCs was developed in order to obtain a large number of fibroblasts and SCs more rapidly.

<table><tbody><tr><td>Alexa Fluor 594 Goat Anti-Mouse IgG(H+L)</td><td>Life Technologies</td><td>A11005</td><td>Dilution: 1:400</td></tr><tr><td>CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG(H+L)</td><td>Proteintech</td><td>SA00013-1</td><td>Dilution: 1:400</td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica Microsystems</td><td>TCS SP5</td><td></td></tr><tr><td>Cell Quest software</td><td>Becton Dickinson Biosciences</td><td></td><td></td></tr><tr><td>D-Hank&#039;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>Fluoroshield Mounting Medium</td><td>Abcam</td><td>ab104135</td><td></td></tr><tr><td>Fixation medium/Permeabilization medium</td><td>Multi Sciences (LIANKE) Biotech, Co., LTD</td><td>GAS005</td><td></td></tr><tr><td>Flow cytometry</td><td>Becton Dickinson Biosciences</td><td>FACS Calibur</td><td></td></tr><tr><td>Mouse IgG1 kappa [MOPC-21] (FITC) - Isotype Control</td><td>Abcam</td><td>ab106163</td><td>Dilution: 1:400</td></tr><tr><td>Mouse monoclonal anti-CD90 antibody</td><td>Abcam</td><td>ab225</td><td>Dilution: 1:1000 for ICC, 0.1 &amp;micro;g for 10&lt;sup&gt;6&lt;/sup&gt; cells for Flow Cyt</td></tr><tr><td>Mouse anti-S100 antibody</td><td>Abcam</td><td>ab212816</td><td>Dilution: 1:400</td></tr><tr><td>Polylysine (PLL)</td><td>Sigma</td><td>P4832</td><td></td></tr><tr><td>Recombinant Human NRG1-beta 1/HRG1-beta 1 EGF Domain Protein</td><td>R&amp;amp;D Systems</td><td>396-HB-050</td><td></td></tr><tr><td>0.25% (w/v) trypsin</td><td>Gibco</td><td>25200-072</td><td></td></tr></tbody></table>

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.

Light microscopic observation 
The SCs and fibroblasts are the two main cell populations obtained in the primary cell culture from nerve tissues. After inoculation for 1 h, most of the cells adhered to the bottom of the dish, and the cell morphology changed from round to oval. After culturing for 24 h, the SCs exhibited a bipolar or tripolar morphology and the length of these ranged from 100 to 200 &#181;m. After 48 h, aggregation and proliferation of cells occurred,...

Watch this Scientific Journal Video about Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro at JoVE.com

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

Here, we present a method to purify 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 ...

purification-fibroblasts-schwann-cells-from-sensory-motor-nerves

Research

JoVE Journal

Neuroscience

7.1K Views.  Nantong University. Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

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

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.

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

Developmental Biology

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Bioengineering

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.

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

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, ...

Enhancing Peripheral Nerve Regeneration with nsPEF-Stimulated Schwann Cells

Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration

Here, we present a protocol for applying nanosecond pulse electric field (nsPEF) to stimulate Schwann cells in vitro. The synthesis and secretion ability of relevant factors and cell behavior changes validated the successful stimulation using nsPEF. The study gives a positive view of the peripheral nerve regeneration method.

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse ...

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.

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

	Autoclave all dissection tools ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Co-culturing of a Dorsal Root Ganglion Explant with Schwann Cells for Neuron Myelination

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 the coculturing model of a dorsal root ganglion explant and Schwann cells. In coculture conditions, ascorbic acid promotes the differentiation of Schwann cells into myelinating forms. Myelinating Schwann cells wrap the axons multiple times to create a myelin sheath, which is essential for nerve function.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Schwann cell culture

	...

Isolation of Schwann Cells from Mouse Spinal Roots

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.

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

Primary Neuronal Culture

Isolation and Culture of Primary Schwann Cells from a Human Dermis

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) ...