Name: purification of fibroblasts and schwann cells from sensory and motor nerves in vitro
Uploaded: 2020-05-20T13:00:00
Description: Watch this Scientific Journal Video about Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro at JoVE.com

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 ...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#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>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 &#181;g for 106 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&#38;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 ...

This protocol can be used to obtain large number of sensor and the motor fibroblast and Schwann cells rapidly. The cells obtained through this message can be used in the study of nerve regeneration and nervous system diseases. We show demonstration can represent the experimental to better control the time of differential digestive step and make the experimental steps easier to understand.To burst use scissors to make a three centimeter incision, in the skin of the back. Remove the spinal column. Carefully open the vertebral canal to expose the spinal cord.Lace the spinal cord in a 60 millimeter Petri dish, with three to four milliliters of ice cold D Hanks balanced salt solution under a dissecting microscope, excise the ventral root and the dorsal root. Place them in ice cold D Hanks balanced salt solution. After removing the HBSS, slice the nerves into three to five millimeter pieces, with scissors.Add one milliliter of 0.25%trypsin and transfer the nerve pieces to five milliliter centrifuge tubes. Incubate at 37 degrees Celsius for 18 to 20 minutes. Next, to stop the digestion, add at three to four milliliters of DMEM containing 10%fetal bovine serum.Pipette the mixture up and down gently about 10 times. Centrifuge at 800 times G for five minutes. After centrifugation discard the supernatant.And re-suspend the precipitate in two to three 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 four to five days at 37 degrees Celsius, in the presence of 5%carbon dioxide.When the culture has reached 90%confluence, wash the cells once using 1XPBS. Then, to digest the Schwann cells, add one milliliter of 0.25%trypsin at 37 degrees Celsius per 60 millimeter dish. After eight to 10 seconds at room temperature, stop the digestion, by adding three milliliters of DMEM supplemented with 10%FBS.To detach the Schwann cells, gently blow on the cell with a pipette. Verify by microscope that the cells are detached. Then collect the medium, which contains the Swan cells, and centrifuge at 800 times G for five minutes.Discard the supernatant, and re-suspend the precipitate in three milliliters of medium. Use this suspension to inoculate uncoated 60 millimeter Petri dishes, and incubate the dishes at 37 degrees Celsius for 30 to 45 minutes. Brands for 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 two days.After removing the medium containing the Schwann cells wash the fibroblasts, adhering to the Petri dishes with 1XPBS. To digest the fibroblasts, add one milliliter of 0.25%trypsin at 37 degrees Celsius. After following digestion to proceed for two minutes at 37 degrees Celsius, end the digestion by adding DMEM supplemented with 10%FBS.To detach the fibroblasts, use a pipette to blow on the bottom of the dish. Collect the medium, including the fibroblasts, and transfer it to centrifuge tubes. Centrifuge the tubes at 800 times G for five minutes.Discard the supernatant, and resuspend the precipitate with two milliliters of DMEM containing 10%FBS. Inoculate the cells in un-coded, 60 millimeter, Petri dishes, and incubate at 37 degrees Celsius for 30 to 45 minutes. Isolate the fibroblasts which will adhere to the Petri dishes, by removing and discarding the growth medium.Add three milliliters of DMEM supplemented with 10%FBS. Incubate at 37 degrees Celsius for two days until the passage one cells reach 90%confluence. Repeat the differential digestion and adherence.After culturing the passage to fibroblasts and Schwann cells for two days, digest the cells, collect them, and count them. Inoculate PLL coated slides at a concentration of one times 10 of these cells per well for ICC staining. Bay's contrast microscopy shows the morphology of cultured Schwann cells and fibroblasts throughout the isolation process.After prolonged culture time, the Schwann cells were clustered together between the fibroblasts or located on the surface of fibroblasts. After digestion for 10 seconds, the Schwann cells, indicated by the red arrows, were round while the fibroblasts remained flat and were attached to the bottom of the dish. After isolation by differential digestion and differential adherence typical cell morphologies were observed for all four cell types.Motor fibroblasts, sensory fibroblasts, motor Schwann cells and sensory Schwann cells. The sensory and motor fibroblasts were visualized using a cone focal laser scanning microscope. The fibroblasts were stained with antibodies against CD 90 X33342 dye was used to label the cell nuclei.The merged images of fibroblasts immunostaining and nuclear staining, indicated that 92.51%and 92.64%of CD 90 and hexed co-labeled cells, were present in the motor and sensory fibroblasts respectively. The sensory and motor Schwann cells were visualized using a confocal laser scanning microscope. The Schwann cells were stained with antibodies against S100 X33342 dye was used to label the cell nuclei.The merge images of Swan cell immunostaining and nuclear staining indicated the presence of 91.61%and 93.56%of S100 and hexed co-labeled cells in motor and sensory Schwann cells, respectively. Cell purity was also analyzed using flow cytometry. In the fibroblast cultures, approximately 90%of cells were fibroblasts, indicated by M2 in the FCA graphs while the remaining cells were Schwann cells.In the Schwann cell cultures, more than 92%of cells were Schwann cells, again indicated by M2 in the FCA graphs, while the remaining cells were fibroblasts. This protocol is rarely useful for starting the mechanism of sensor and motor nerve regeneration of fibroblasts, and the Schwann cells transplantation, to promote nerve regeneration.

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