co-culture of schwann cells with drg neurons on a pre-stretched membrane

Co-Culture of Schwann Cells with DRG Neurons on a Pre-Stretched Membrane

Prior to seeding the DRG neurons, remove the PLL solution from the chamber. Rinse the PDMS surface with sterile water and air-dry it. After resuspending the cells, let the suspension sit for 1 to 2 minutes to allow the debris to settle to the bottom of the tube. Next, add 1.5 milliliters of cell suspension into each stretch chamber, and incubate it at 37 degrees Celsius with 5% CO2.
To eliminate glial cells at one day in vitro, add 10 microliters or 15 microliters of the FdU/U mixture stock solution to each well. After 7 hours, replace this medium with fresh standard growth medium, and place the pre-stretched culture device in a 37 degrees Celsius incubator in 5% CO2. During the cell culture period, change the medium every two days by replacing half the spent medium with fresh medium.
In this procedure, remove the culture medium from the Schwann cells, and add 5 milliliters of 0.05% trypsin-EDTA into the flask. Incubate the cells at 37 degrees Celsius in 5% CO2 for 2 to 3 minutes. Check the cells under the optical microscope using a 10x objective to see if they lift up from the flask. Then, add 5 milliliters of culture medium to the cells in the flask, and mix.
Subsequently, add the cell suspension to a 15-milliliter centrifuge tube and centrifuge at 20 degrees Celsius for 5 minutes. Then, remove the supernatant and resuspend the pellet in 7 milliliters of standard DOG growth medium. Following this, transfer 10 microliters of the cell suspension into a 0.5-milliliter microcentrifuge tube. Mix it with 10 microliters of trypan blue, and then count the cell number with a hemocytometer, using a 10x objective.
Add the growth medium to dilute the cell suspension to 5,000 cells per milliliter. Next, remove 0.5 milliliters of the medium from the DRG culture in the stretched chamber, and add 0.5 milliliters of Schwann cell suspension to the DRG culture. Culture the cells for one week at 37 degrees Celsius in 5% CO2. Change the medium every two days by replacing half of the spent medium with fresh standard growth medium.

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1. Culture of dorsal root ganglion (DRG) on Pre-stretched Surface
<ol>
	<li>Preparation of Pre-stretched Anisotropic Surface
	<ol>
		<li>Mix a 10:1 solution of base and curing agent and pour the mixture into a tissue culture dish (12 cm diameter). Use 4,900 mg base and 490 mg curing agent for the total crosslinking mixture.</li>
		<li>Keep the gel mixture under vacuum for 20 min to remove air bubbles.</li>
		<li>Place the gel mixture in the oven for overnight curing at 60 &#176;C.</li>
		<li>After the poly-dimethyl-siloxane (PDMS) membrane cures, treat the surface with oxygen plasma using a plasma cleaning/etching system for 3 min at 165 mTorr and 65 sccm flow of O2.</li>
		<li>Cut a piece of rectangular membrane (5 &#215; 3.5 cm) from the dish; while being cognizant of which side is oxygen-treated, make sure the oxygen-treated side is facing up and fix it onto a stretching frame. Place the frame on a stretching stage and evenly stretch by turning the knob on the stage until it reaches 10% elongation (or some other pre-determined stretch; the elongation can be directly read from the stretching stage) in the longer axis. Fix the stretch by tightening the screws on the frame.</li>
		<li>Remove the frame from the stage. Ensure the membrane surface is dry and free of dust, then place a silicone chamber onto the membrane, allowing the sticky side of the chamber to attach tightly to the membrane. 
		&#8203;Note: The silicone chamber provides a well for retaining the cell medium on the PDMS membrane. The device design is shown in Figure 1.</li>
		<li>Sterilize the surface with UV for 10 min.</li>
		<li>Add 1 ml Poly-L-Lysine (PLL) (0.01% in Phosphate Buffered Saline) to the PDMS surface within the chamber within 6 hr of plasma treatment and incubate for 2 hr at 37 &#176;C prior to seeding the DRG neurons. This enhances cell attachment.</li>
	</ol>
	</li>
	<li>&#160;Prior to seeding the cells, remove the PLL solutions from the chamber and rinse the PDMS surface with sterile water and air dry.</li>
	<li>Take a suspension of the DRG cells. Let the suspension set for 1 - 2 min to allow the debris to settle to the bottom of the tube. Add 1.5 ml of cell suspension into each stretch chamber and incubate at 37 &#176;C at 5% CO2.</li>
	<li>To eliminate glial cells, at 1 day in vitro (DIV), add 10 &#181;l (or 15 &#181;l) mixture of fluoro-2 deoxy-uridine and uridine (FDU-U) stock solution (See Table of Materials) to each well. After 7 hr, replace this medium with fresh standard growth medium and place the pre-stretched culture device in a 37 &#176;C incubator with 5% CO2.</li>
	<li>During the cell culture period (2 - 3 weeks), change the media every two days by replacing half the spent medium with fresh medium. 
	Note: After culturing on the stretched and unstretched surfaces for 2 weeks, purified SCs are added to the chamber and cultured for another week (step 2.7).</li>
</ol>
2. Co-culture of Schwann Cells (SCs) with DRG Neurons on Pre-stretched Surface
<ol>
	<li>Remove the culture medium from the flak containing SCs, and add 5 ml 0.05% Trypsin-EDTA into the flask. Incubate the cells at 37 &#176;C at 5% CO2 for 2 - 3 min.</li>
	<li>Check cells under the optical microscope using a 10X objective to see if they lift up from the flask, then add 5 ml of culture medium and mix with the cells in the flask.</li>
	<li>&#160;Add the cell suspension to a 15 ml centrifuge tube and centrifuge at 200 x g and 20 &#176;C for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 7 ml standard DRG growth media.</li>
	<li>Take 10 &#181;l of cell suspension into a 0.5 ml microcentrifuge tube, mix with 10 &#181;l of trypan blue, and then count the cell number using a hemocytometer under optical microscopy using a 10X objective. Dilute the cell suspension to 5,000 cells per ml by adding DRG growth media.</li>
	<li>Remove 0.5 ml of medium from the DRG culture in the stretched chamber and add 0.5 ml of SC suspension to the DRG culture.</li>
	<li>Culture the cells for 1 week at 37 &#176;C and 5% CO2. Change the media every two days by replacing half the spent medium with fresh standard growth medium for DRG for 1 week.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium 1X</td>
			<td>GibcoBRL</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement 50X</td>
			<td>GibcoBRL</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax-I 100X</td>
			<td>GibcoBRL</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumax-I</td>
			<td>GibcoBRL</td>
			<td>11020-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve Growth Factor-7S</td>
			<td>Invitrogen</td>
			<td>13290-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>GibcoBRL</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA/1mM EDTA</td>
			<td>GibcoBRL</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Trevigen</td>
			<td>3438-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>p-6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-2 deoxy-uridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Isolation Buffer</td>
		</tr>
		<tr>
			<td>Type I Collagenase</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone chamber</td>
			<td>Greiner bio-one</td>
			<td>FlexiPERM ConA</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaning/etching system</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard growth medium</td>
			<td>&#160;&#160;</td>
			<td>&#160; &#160;</td>
			<td>For 500 ml Neurobasal Medium 1X, add 10 ml of B-27 50X, 5 ml of Glutamax-I 100X,&#160;2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 &#956;l of NGF-- 7S (50 ug/ml).</td>
		</tr>
		<tr>
			<td>FDU and Uridine stock solution</td>
			<td></td>
			<td></td>
			<td>FDU 100mg in 10ml of ddw (10mg/ml), filter in the hood and divided in 500ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Uridine 5g in 166.7ml of ddw (33mg/ml), filter in hood, divide in 200ul aliquots and store at -20 &#186;C.</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Take 61.5ul of FDU (10mg/ml) and 20.5ul of Uridine(33mg/ml), and add 4918ul of ddw to a final stock concentration,&#160;then divide in 1 ml aliquots and store at -20 &#186;C.</td>
		</tr>
	</tbody>
</table>

Source: Liu, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54085">An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the co-culturing of dorsal root ganglion (DRG) neurons with Schwann cells. DRG neurons are first grown on a pre-stretched membrane for aligned axon growth. Schwann cells are then introduced, which attach to the axons, insulating them and promoting neural cell survival and function.

<img alt="Figure 1" src="/files/ftp_upload/22548/22548_Figure1.jpg" /> 
Figure 1: Schematic for the Pre-stretch Procedure. (a) A piece of PDMS membrane is clipped onto two strips of the frame that are slidable. The frame of the device is approximately 7&#34; x 5&#34;; the braces are about 5&#34; x 1; the clamps on the braces are about 3&#34; x .25&#34;, and the cup is about 1.5&#34; diameter wide open top, 1&#34; open bottom, and about 0.25&#34; tall. (b) Place the frame onto the stretching stage, turn the roller to apply 10% elongation, and then fix the positions of the strips by tightening the screws. (c) Remove the frame from the stretching stage, and attach a silicon chamber onto the membrane.

1. Culture of dorsal root ganglion (DRG) on Pre-stretched Surface

	Preparation of Pre-stretched Anisotropic Surface
	
		Mix a 10:1 solution of base and ...

Take a silicon chamber attached to a matrix-coated, stretched silicon membrane.
Add dorsal root ganglia or DRG cell suspension and incubate.
The matrix promotes cell attachment and multiplication, while the stretched surface guides axon alignment.
Add growth inhibitors that eliminate actively dividing glial cells, allowing non-dividing neurons to survive.&#160;
Remove the medium. Add a growth medium and incubate.
The neuron senses an increased membrane stiffness in the stretched direction and orients the axon growth accordingly.&#160;
Add cultured and harvested Schwann cells to the DRG culture and incubate.
The Schwann cells attach to the regenerated axon, insulating the neuron.
This enhances the survival and functionality of neurons, establishing a co-culture.

83 조회수. Pre-Stretched Membrane에서 Schwann 세포와 DRG 뉴런의 공동 배양

비디오: Pre-Stretched Membrane에서 Schwann 세포와 DRG 뉴런의 공동 배양 - 실험

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

신경 전달 물질 방출 공부 하 느 루트 중추 절연 및 주 문화

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

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JoVE Journal

신경과학

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.

쥐 등쪽 뿌리 신경절 외식편과 슈반 세포의 공동 배양에서 말초 축삭의 시험관 내 수초화

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

면역패닝에 의한 성체 마우스 배근 신경절 뉴런 배양의 농축

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...

Co-Culture of Schwann Cells with DRG Neurons on a Pre-Stretched Membrane

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Co-Culture of Schwann Cells with DRG Neurons on a Pre-Stretched Membrane