co-culturing of a dorsal root ganglion explant with schwann cells for neuron myelination

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

Take a four-well plate containing 190 microliters of DRG growth medium in each well, and carefully transfer a single DRG into the center of each well using fine forceps and a spatula. Then, place the DRG explant cultures in the incubator at 37 degrees Celsius and 5% carbon dioxide. The next day, carefully add 50 microliters of the DRG growth medium to each well, and observe DRG explant adherence and axon outgrowth using a microscope daily. For co-culturing on day three of the DRG explant culture, carefully replaced the DRG growth medium with 250 microliters of the co-culture medium containing 30,000 Schwann cells per well.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
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&#160;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>Sciatic nerve preparation 
	NOTE: All sciatic nerve preparation steps are performed under a clean bench.
	<ol>
		<li>Prepare one 100 mm TC dish with 5 mL of ice-cold Dulbecco&#39;s phosphate-buffered saline (DPBS) without Ca2+&#160;and Mg2+, one 100 mm TC dish with 5 mL of ice-cold Leibovitz&#39;s L-15 medium, and one 100 mm TC dish with 5 mL of ice-cold Leibovitz&#39;s L-15 medium and 50 &#181;g/mL gentamycin.</li>
		<li>Clean all the instruments by autoclaving. Spray the instruments and working area with 70% ethanol.</li>
		<li>Euthanize five 3-week-old male Sprague Dawley rats using CO2&#160;inhalation and decapitation. Spray the rat&#39;s torso with 70% ethanol.</li>
		<li>Open the dorsal lower left limb with scissors and remove the biceps femoris muscle carefully. Loosen the sciatic nerve by smooth elevation with curved forceps, ensuring not to bruise the nerve.</li>
		<li>Hold the most proximal part of the nerve with the curved forceps to straighten the nerve, and clip the nerve as high as possible using scissors. Then, clip the nerve close to the sacral plexus and the paw with scissors. Repeat steps 1.3.4-1.3.5 for the right side. 
		NOTE: While opening the limb, take care not to bruise any blood vessels.</li>
		<li>Using forceps, put the left and right sciatic nerves into a 100 mm TC dish with ice-cold DPBS.</li>
	</ol>
	</li>
	<li>Sciatic nerve refurbishment
	<ol>
		<li>Use forceps to transfer all the nerves to a 100 mm TC dish with ice-cold Leibovitz&#39;s L-15 medium and 50 &#181;g/mL gentamycin. Continue using a stereomicroscope and remove fat, muscle, and blood vessels from the nerves with two pairs of fine forceps. Grab the nerves with forceps and transfer them to a 100 mm TC dish with ice-cold Leibovitz&#39;s L-15 medium.</li>
		<li>Identify the proximal and distal ends of the sciatic nerve. Remove the epineurium with one pair of fine forceps in a proximal to distal direction, while holding the proximal nerve end with the second pair of fine forceps.</li>
		<li>Transfer the purified nerves to a 100 mm TC dish with ice-cold Leibovitz&#39;s L-15 medium and 50 &#181;g/mL gentamycin. Tease the isolated nerve fascicles to separate and isolate single nerve fibers using two pairs of fine forceps.</li>
		<li>Transfer the nerve fibers to a 50 mL tube using a 10 mL serological pipette and take up as little medium as possible. Add 50 mL of Leibovitz&#39;s L-15 medium with 50 &#181;g/mL gentamycin to the nerve fibers and slew a few times in the 50 mL tube.</li>
	</ol>
	</li>
	<li>Enzymatic digestion of the sciatic nerve&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	&#8203;NOTE: The next steps (steps 1.5-1.8, 2.1, and 2.2) 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&#160;g&#160;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 TC 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&#160;for 18 h, and stop the digestion by adding 10 mL of 40% FCS in Hanks&#39; balanced salt solution, without Ca2+&#160;and Mg2+&#160;(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&#160;g&#160;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&#160;g&#160;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&#160;day, and observe the cell appearance and confluency using a microscope. 
		&#8203;NOTE: Schwann cells and DRG explants need to be prepared in a timely, coordinated manner for the coculture. Make sure a rat with embryos at E 13.5 is available for DRG preparation when the Schwann cells are close to a confluency of 80%.</li>
	</ol>
	</li>
	<li>Cell trypsinization and magnetic separation&#160;&#160;&#160; 
	NOTE: For exemplary pictures of different Schwann cell culture stages, see&#160;Figure 1.
	<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 (ethylenediaminetetraacetic acid) (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.&#160;&#160;&#160;&#160; 
		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&#160;g&#160;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&#160;cells. Add 10 &#181;L of Thy-1 microbeads per 1 x 107&#160;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&#160;g&#160;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&#160;g&#160;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 (see step 3.1.1). Count the cells after staining with trypan blue and a staining chamber.</li>
	</ol>
	</li>
</ol>
2. DRG explant culture
<ol>
	<li>DRG growth medium preparation
	<ol>
		<li>Prepare DRG growth medium by adding 2% B27, 2% horse serum, 1% L-glutamine, 0.5% penicillin/streptomycin, and 10 ng/mL nerve growth factor (NGF) to the neurobasal medium. Store the growth medium at 4 &#176;C. 
		NOTE: The growth medium can be used for 2 days.</li>
	</ol>
	</li>
	<li>Coating for DRG explants
	<ol>
		<li>Incubate the coverslips in 70% ethanol for 1 h and place in the wells of 4-well dishes using curved forceps. After the ethanol has dried, apply 300 &#181;L of 0.2 mg/mL poly-D-lysine (PDL) per well and incubate overnight at 37 &#176;C and 5% CO2.</li>
		<li>Wash the coverslips 3x for 5 min each with DPBS consecutively. Take off the DPBS, apply 300 &#181;L of 1 &#181;g/mL laminin to the coverslips, and incubate overnight at 37 &#176;C and 5% CO2.</li>
		<li>After three washing steps with DPBS for 5 min, replace the DPBS with 190 &#181;L of DRG growth medium. Place the 4-well plates into the incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>DRG preparation&#160; 
	NOTE: Harvest the DRG of embryonic rats under a clean bench.
	<ol>
		<li>Before preparation, clean all the instruments with 70% ethanol. Fill 10 (two per embryo) 35 mm TC dishes with 2 mL of ice-cold HBSS per dish, and two 100 mm TC dishes with 5 mL of ice-cold HBSS per dish.</li>
		<li>Euthanize the pregnant rats (adult female Sprague Dawley rats, E13.5) by CO2&#160;inhalation and decapitation.</li>
		<li>Spray the body with 70% ethanol and open the ventral torso of the rat. Carefully remove the uterus and place it into a 100 mm TC dish with ice-cold HBSS.</li>
		<li>Hold the uterus using curved forceps and open the uterus wall with fine forceps. Remove one amniotic sac and open it carefully by pinching a hole with fine forceps.</li>
		<li>Remove the embryo from the surrounding tissues, cut the umbilical cord, and decapitate the embryo using fine forceps. Place the torso into a 100 mm TC dish filled with HBSS using curved forceps and a spatula.</li>
		<li>Quickly remove all the embryos from the uterus and transfer them into one 100 mm TC dish filled with HBSS. If there are more than five embryos, prepare an additional 35 mm TC dish with 2 mL of HBSS, according to step 2.3.1.</li>
		<li>Gently place one embryo torso into a 35 mm TC dish filled with HBSS using a spatula and curved forceps. Under a stereomicroscope, open the dorsal part of the torso to divide the embryo into two halves using fine forceps and micro scissors. Turn one half to the side and identify the strand of DRG located in a line at the dorsal part of the embryo.</li>
		<li>Cut out the DRG as a whole strand using fine forceps and micro scissors. Place the DRG in a fresh 35 mm TC dish filled with 2 mL of HBSS, and separate a single DRG from the remaining tissue using fine forceps and micro scissors.</li>
	</ol>
	</li>
	<li>DRG cell culture
	<ol>
		<li>Take 4-well plates containing 190 &#181;L of DRG growth medium from the incubator to the clean bench where the DRG are to be prepared. Transfer a single DRG carefully into one well of a 4-well culture plate using fine forceps and a spatula. Place the DRG into the center of each well, since a central position is important for the attachment of the DRG.</li>
		<li>Work under sterile conditions from now on. Place the explant cultures in the incubator at 37 &#176;C and 5% CO2. The next day, add 50 &#181;L of the DRG growth medium carefully to each well using a 100 mL pipette.</li>
		<li>Observe DRG explant adherence and axon outgrowth using a microscope daily, and discard DRG explants that have detached from the coverslip or failed to outgrow axons on day 3 of culture. 
		&#8203;NOTE: DRG explants are very fragile in the first days of culture and need to be handled with care, especially when moving the plates in and out of the incubator when the medium is changed, or even when closing the incubator door. The precise volume of 190 &#181;L of medium per well is crucial to keep the DRG explants in place during the first day of culture. Loose DRG explants can be identified easily during the daily control, as they swim in the medium instead of adhering to the coverslip.</li>
	</ol>
	</li>
</ol>
3. Coculture
<ol>
	<li>Transfer of Schwann cells to the DRG explant culture
	<ol>
		<li>Prepare the coculture medium by adding 0.1% ascorbic acid to the DRG growth medium.</li>
		<li>On day 3 of the DRG explant culture, carefully replace the DRG growth medium with 250 &#181;L of the coculture medium containing 30,000 Schwann cells (from step 1.8) per well.</li>
		<li>Keep the coculture of the DRG explants and Schwann cells for up to 22 days. Replace 250 &#181;L of the coculture medium carefully every other day, and observe the appearance of the cells using a microscope. 
		NOTE: For an example of DRG axons and Schwann cells in the coculture in the first days of culture, see&#160;Figure 2.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ascorbic acid&#160;</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>A4403-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>B27-supplement</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tip, 100 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>70760212</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tip, 1250 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>701186210</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tip, 20 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>701114210</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tip, 300 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>70765210</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Carl Roth, Karlsruhe, Germany&#160;</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&#160;Incubator Heracell</td>
			<td>Heraeus Instruments, Hanau, Germany&#160;</td>
			<td>51017865</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 12 mm</td>
			<td>Carl Roth, Karlsruhe, Germany&#160;</td>
			<td>P231.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved fine forceps&#160;</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11370-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water (Water Purification System)&#160;</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&#160;</td>
			<td>31331093</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (no Ca2+&#160;and no Mg2+)</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>D8537-6X500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>VWR, Radnor, USA&#160;</td>
			<td>1009862500</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>F7524</td>
			<td>FCS must be tested for Schwann cell culture</td>
		</tr>
		<tr>
			<td>Fine forceps (Dumont #5)</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>F6886-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany</td>
			<td>5710064</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (no Ca2+&#160;and no Mg2+)&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>14170138</td>
			<td></td>
		</tr>
		<tr>
			<td>HERAcell Incubator</td>
			<td>Heraeus Instruments, Hanau, Germany&#160;</td>
			<td>51017865</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraguard ECO 1.2</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>51029882</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Pan-Biotech, Aidenbach, Germany</td>
			<td>P30-0712</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#180;s L-15 Medium</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Multistand&#160;</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&#160;</td>
			<td>Motic, Wetzlar, Germany</td>
			<td>Motic BA 400</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Axio observer 7</td>
			<td>Zeiss, Oberkochen, Germany&#160;</td>
			<td>491917-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>VWR, Radnor, USA&#160;</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&#160;</td>
			<td>Assistant, Erlangen, Germany</td>
			<td>40441 &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuregulin</td>
			<td>Peprotech, Rocky Hill, USA</td>
			<td>100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>N1408</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Biozol, Eching, Germany</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon multidishes, 4 well</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>D6789</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics, New Jersey, USA&#160;</td>
			<td>10342243</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetboy</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>4430000018&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>2231300004</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>P4707-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes, 15&#160;mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes, 50&#160;mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>62547254</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction vessels, 1.5 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>72690001</td>
			<td></td>
		</tr>
		<tr>
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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.

<img alt="Figure 1" src="/files/ftp_upload/22596/22596_Figure 1.JPG" /> 
Figure 1: Stages of Schwann cell culture. Exemplary brightfield pictures of cultured Schwann cells (A) before and (B) after magnetic cell separation. Before magnetic cell separation, the culture includes Schwann cells with an elongated and spindle shape morphology, flat and spread-out appearing fibroblasts, and remnants of connective tissue. To achieve a purer culture of Schwann...

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

	...

Begin with a multi-well plate containing a coverslip coated with poly-D-lysine and a neuron growth medium.
Place the mouse embryonic dorsal root ganglion, or DRG, rich in sensory neurons, into this well and incubate.
DRG neurons utilize the nutrients and growth factors that facilitate their growth, followed by the extension of neuronal projections, forming axons.
These axons grow out from DRG tissue, establishing the DRG explant culture.
Replace the medium with an ascorbic acid-supplemented co-culture medium with Schwann cells, a specialized glial cell.
Over time, axons secrete signaling molecules that trigger Schwann cell migration toward the axons.
Meanwhile, the ascorbic acid stimulates various signaling pathways in the Schwann cells.
These activated cells begin to extend their lipid-rich plasma membranes around the axons, wrapping them in multiple layers to form the myelin sheath.
This myelination results in an insulating covering around the axons, which is essential for efficient neuronal communication.

45 조회수. Neuron Myelination을 위한 Schwann Cells와 Dorsal Root Ganglion Explant의 공동 배양

비디오: Neuron Myelination을 위한 Schwann Cells와 Dorsal Root Ganglion Explant의 공동 배양 - 실험

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

루트 신경절 신경 세포와 차별화 된 지방 유래 줄기 세포를 이룬다 :<em&gt; 체외</em&gt; 공동 문화 모델은 주변 신경 재생을 연구하는

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

연구

JoVE Journal

신경과학

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

리소 레시틴의 초점 주사 실험 탈수 초화 및 설치류 척수의 재유 수화

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

접근 방법은 등쪽 뿌리 신경절 뉴런의 정렬 및 수초를 강화하는

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

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

내레이션 대본

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