Name: Video: Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies - Concept
Uploaded: 2023-04-30T14:49:02.000+00:00
Duration: 1 min 40 s
Description: 1.4K Views. Source: Eldeiry, M. et al. Spinal Cord Neurons Isolation and Culture from Neonatal Mice. J. Vis. Exp. (2017) In this video we describe a step-by-step protocol to isolate spinal cord neurons from a neonatal mouse for in vitro studies.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Harvesting Spinal Cords
NOTE: All instruments should be autoclaved (135 &#176;C and 30 psi for 4 x 7 min cycles) for sterility.
<ol>
	<li>Euthanize 1&#8211;3 day-old C57BL/6 mouse pups in a chamber with isoflurane. Wait 30 s after the cessation of movement and pinch the leg to confirm a lack of response.</li>
	<li>Separate the head from the body using scissors, with pup in the prone position.</li>
	<li>Stabilize the hind legs or tail and arms on the procedure table, with dorsal side facing the user.</li>
	<li>Cut the skin off using curved iris scissors.</li>
	<li>Cut the spinal cord from the lumbar region just above the hips and proceed to cut both sides of the thorax to separate it from the body. 
	NOTE: This requires the careful dissection of the spinal cord from visceral organs to avoid inadvertent damage to other organs (Figure 1a).</li>
	<li>Wash sequentially for 10 s in 3 x 10 cm Petri dishes containing 5 mL of 0.2 &#956;m filter-sterilized Phosphate-Buffered Saline (PBS) to remove excess tissue.</li>
	<li>Insert a 22G needle and syringe filled with 5 mL of filter-sterilized PBS into the caudal end of the spinal column and flush cranially, allowing the cord to exit into a fourth Petri dish (Figure 1b).</li>
	<li>Collect the spinal cord in a 15 mL tube with 5 mL of HABG (Table 1) on ice. Use care to avoid crushing the spinal cord.</li>
	<li>Repeat steps 1.1&#8211;1.8 for each pup in the litter. 
	NOTE: Ideally, this process should take less than 30 min per spinal cord to ensure healthy neuron isolation.</li>
</ol>
2. Isolating Neurons
NOTE: The following step should be performed in a laminar flow hood. Familiarity with basic sterile technique is expected.
<ol>
	<li>Tissue Mincing
	<ol>
		<li>Take the tube containing the spinal cords and shake lightly to suspend the tissue.</li>
		<li>Pour tissue from the tube into a 60 mm glass Petri dish and dice with a razor blade to create fine pieces ~0.5 mm in size.</li>
		<li>Transfer the tissue with a wide-bore pipette into a 15 mL tube containing 5 mL of HABG.</li>
		<li>Place it in a 30 &#176;C water bath for 30 min to allow the cells to equilibrate at this temperature. Keep the cells on a shaker just enough to allow them to be suspended in the fluid. 
		NOTE: This step is done to avoid shocking the cells upon transfer from ice to the digestion medium. Keeping the cells at 30 &#176;C helps to decrease cell death associated with an otherwise increased metabolism at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the digestion medium (Table 1).</li>
	<li>Prepare the density gradient (Table 1).
	<ol>
		<li>Prepare each of the 4 layers in 4 separate 15 mL tubes, as outlined in&#160;Table 1.</li>
		<li>Add 1 mL from each layer into a new 15 mL tube. Start with layer 1 at the bottom and sequentially add until reaching layer 4 at the top. Avoid disturbing the layers while adding.</li>
	</ol>
	</li>
	<li>Wash the PDL-coated plates. Wash with sterile water for a few minutes before removing any remaining water and allowing them to dry for 1 h.</li>
	<li>Transfer the tissue to digestion medium.
	<ol>
		<li>Remove the tissue-containing tube from the shaker water bath at 30 &#176;C and allow it to settle for a few minutes.</li>
		<li>Remove the digestion medium tube from the 37 &#176;C water bath and aspirate it into a leur-lock syringe.</li>
		<li>Aspirate off the excess HABG from the tissue-containing tube.</li>
		<li>Use a leur-lock 0.2 &#956;m filter on the syringe to add digestion medium to the tissue-containing tube.</li>
		<li>Place the tube in a 30 &#176;C water bath for 30 min. Keep the cells shaking just enough to allow them to be suspended in the fluid. 
		NOTE: It is important not to keep the cells in the digestion medium for too long or to let the temperature get too high, which could lead to excessive digestion and result in the tissue becoming suspended in a gelatinous mixture.</li>
	</ol>
	</li>
	<li>During this period, coat the laminin.</li>
	<li>Perform trituration (i.e.&#160;separating the cells from the tissue).
	<ol>
		<li>Remove the tube from the shaking 30 &#176;C water bath and allow it to settle for a few min.</li>
		<li>Aspirate excess digestion medium.</li>
		<li>Suspend the tissue in 2 mL of HABG.</li>
		<li>Using a narrow-bore pipette, triturate 10x for 45 s. 
		NOTE: This is probably the single most crucial step and can significantly decrease the yield if not done properly.
		<ol>
			<li>Aspirate the tissue into the pipette and immediately empty the contents back.</li>
			<li>Avoid introducing air, as it will significantly decrease the viable yield. 
			NOTE: The ideal pipette is a 9&#34; glass pipette. The tip of the pipette should be fire polished to smooth out rough surfaces. It should then be siliconized by placement in a 1:20 solution of dichlorodimethylsilane (DMDCS) in chloroform and left O/N. The pipette should then be removed and allowed to air dry. Subsequently, it should be autoclaved for sterilization. 
			CAUTION: DMDCS and chloroform are highly flammable, and siliconizing should be carried out in a fume hood.</li>
		</ol>
		</li>
		<li>Aspirate the top 2 mL of supernatant and place it into a new 15 mL tube labeled &#34;collection&#34;.</li>
		<li>Repeat steps 2.7.3&#8211;2.7.5 two additional times (the cell collection tube should have 6 mL by the end).</li>
		<li>Slowly transfer the collection tube contents into the gradient tube prepared in step 2.2, avoiding the disruption of the gradient.</li>
	</ol>
	</li>
	<li>At this point, remove the previously prepared neurobasal medium (Table 1) from the refrigerator and allow it to warm in a 37 &#176;C bath.</li>
	<li>Purify the neurons.
	<ol>
		<li>Centrifuge the gradient tube for 15 min at 800 x g and 22 &#176;C.</li>
		<li>Collect the desired layer(s) with a pipette (Figure 2) and place in a new 15 mL tube. For the highest-purity neuron isolation (i.e.&#160;&#62;90%), collect layer 3. For more yield with less purity (i.e.&#160;&#62;70&#8211;80%), collect layers 2 &#38; 3.</li>
		<li>Dilute out the density gradient by adding 5 mL of HABG to the newly collected layers.</li>
		<li>Centrifuge at 200 x g for 2 min at 22 &#176;C.</li>
		<li>Discard the supernatant, resuspend in 5 mL of HABG, and flick the pellet to suspend the cells.</li>
		<li>Centrifuge at 200 x g for 2 min at 22 &#176;C.</li>
		<li>Discard the supernatant, resuspend in 3 mL of neurobasal medium, and flick the pellet to resuspend the cells.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media</td>
			<td>Storage</td>
			<td colspan="3">Ingredients</td>
			<td>Preparation</td>
		</tr>
		<tr>
			<td rowspan="4">HABG</td>
			<td rowspan="4">4 &#176;C/24h</td>
			<td></td>
			<td colspan="2">100 mL</td>
			<td rowspan="4">- Thaw B27 in 4 &#176;C fridge O/N 
			- Filter sterilize (0.22 &#181;m)</td>
		</tr>
		<tr>
			<td>Hibernate A</td>
			<td colspan="2">97.8 mL</td>
		</tr>
		<tr>
			<td>B27 [2%]</td>
			<td colspan="2">2 mL</td>
		</tr>
		<tr>
			<td>GlutaMAX [0.5 mM]</td>
			<td colspan="2">0.25 mL</td>
		</tr>
		<tr>
			<td rowspan="5">Neurobasal Media</td>
			<td rowspan="5">4 &#176;C/1 week</td>
			<td></td>
			<td colspan="2">100 mL</td>
			<td rowspan="5">- Thaw B27 in 4 &#176;C fridge O/N 
			- Filter sterilize (0.22 &#181;m) 
			-&#160;Aliquot 50 mL portions into 50 mL tubes (store at 4 &#176;C)</td>
		</tr>
		<tr>
			<td>Neurobasal A</td>
			<td colspan="2">97 mL</td>
		</tr>
		<tr>
			<td>B27 [2%]</td>
			<td colspan="2">2 mL</td>
		</tr>
		<tr>
			<td>GlutaMAX [0.5 mM]</td>
			<td colspan="2">0.25 mL</td>
		</tr>
		<tr>
			<td>Pen/Strep [1%]</td>
			<td colspan="2">1 mL</td>
		</tr>
		<tr>
			<td rowspan="4">Digestion Media</td>
			<td rowspan="4">Day of Isolation</td>
			<td></td>
			<td colspan="2">10 mL</td>
			<td rowspan="4">- Shake vigorously 
			- Let it sit in 37&#176;C bath for 30 min (ideally prepared 30 min prior to use) 
			- Filter sterilize (0.22 &#181;m)</td>
		</tr>
		<tr>
			<td>HA-Ca</td>
			<td colspan="2">10 mL</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td colspan="2">25 mg</td>
		</tr>
		<tr>
			<td>GlutaMAX [0.5 mM]</td>
			<td colspan="2">0.025 mL</td>
		</tr>
		<tr>
			<td rowspan="7">Density Gradient</td>
			<td rowspan="7">Day of Isolation</td>
			<td></td>
			<td colspan="2">1 Gradient</td>
			<td rowspan="7"></td>
		</tr>
		<tr>
			<td rowspan="2">Layer</td>
			<td>OptiPrep</td>
			<td>HABG</td>
		</tr>
		<tr>
			<td>(0.13 mL)</td>
			<td>(5.29 mL)</td>
		</tr>
		<tr>
			<td>1 Bottom</td>
			<td>0.26 mL</td>
			<td>1.24 mL</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.19 mL</td>
			<td>1.31 mL</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.15 mL</td>
			<td>1.35 mL</td>
		</tr>
		<tr>
			<td>4 Top</td>
			<td>0.11 mL</td>
			<td>1.39 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Preparation of the media and solutions used in this protocol.

<table><tbody><tr><td>Hibernate A Medium - 500 mL</td><td>Thermo-Fisher</td><td>A1247501</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/A1247501&lt;/a&gt;</td></tr><tr><td>Hibernate A Minus Calcium - 500 mL</td><td>Brainbits</td><td>HA-Ca</td><td>&lt;a href=&quot;http://www.brainbitsllc.com/&quot;&gt;http://www.brainbitsllc.com/ hibernate-a-minus-calcium/&lt;/a&gt;</td></tr><tr><td>Glutamax 100X - 100 mL</td><td>Thermo-Fisher</td><td>35050061</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/35050079&lt;/a&gt;</td></tr><tr><td>B27 Supplement 50X - 10 mL</td><td>Thermo-Fisher</td><td>17504044</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/17504044&lt;/a&gt;</td></tr><tr><td>Papain, Lyophilized - 100 mg</td><td>Worthington</td><td>LS003119</td><td>https://www.worthington-biochem.com/PAP/cat.html</td></tr><tr><td>Neurobasal A Medium - 500 mL</td><td>Thermo-Fisher</td><td>10888022</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/10888022&lt;/a&gt;</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ mL)</td><td>Thermo-Fisher</td><td>15140122</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/15140122&lt;/a&gt;</td></tr><tr><td>Poly-D-Lysine (PDL) hydrobromide- 5 mg</td><td>Sigma-Aldrich</td><td>P6407-5MG</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/p6407? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Mouse Laminin - 1 mg</td><td>Thermo-Fisher</td><td>23017015</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/23017015&lt;/a&gt;</td></tr><tr><td>Trypan Blue - 20 mL</td><td>Sigma-Aldrich</td><td>T8154-20ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/t8154? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>OptiPrep Density Gradient Medium-250 mL</td><td>Sigma-Aldrich</td><td>D1556-250ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/d1556? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Dichlorodimethylsilane (DMDCS, Sigma Silicoat)</td><td>Sigma-Aldrich</td><td>440272-100ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/aldrich/440272? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>288306-1L</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sial/288306? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Glass Pippette - 9&quot;</td><td>Sigma-Aldrich</td><td>13-678-20C</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/cls7095d9? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Pipette bulb - 5 mL</td><td>Sigma-Aldrich</td><td>Z186678-3EA</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/aldrich/z186678? lang=en&amp;amp;region=US&amp;amp;cm_sp=Insite-&lt;br/&gt; _-prodRecCold_xviews-_- prodRecCold10-1&lt;/a&gt;</td></tr><tr><td>BRAND&lt;sup&gt;&amp;reg;&lt;/sup&gt; Petri dish, glass - 60 x 15 mm</td><td>Sigma-Aldrich</td><td>BR455717-10EA</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/aldrich/br455717? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Sterile 24-Well Cell Culture Plate</td><td>Sigma-Aldrich</td><td>M8812-100EA</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/m8812? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr></tbody></table>

spinal cord neuron isolation: a density gradient procedure to isolate neonatal spinal cord neurons for in vitro studies

Source: Eldeiry, M. et al. <a href="https://www.jove.com/video/55856" target="_blank">Spinal Cord Neurons Isolation and Culture from Neonatal Mice.</a> J. Vis. Exp. (2017)
In this video we describe a step-by-step protocol to isolate spinal cord neurons from a neonatal mouse for in vitro studies.

<img alt="Figure 1" src="/files/ftp_upload/20698/20698_Figure1.jpg" /> 
Figure 1: Spinal column dissected and spinal cord released from a 3 day-old neonatal mouse. The arrow in (a) points towards the caudal end of the spine, where a needle is inserted to release the spinal cord. A released spinal cord (b) is shown submerged in PBS.
<img alt="Figure 1" src="/files/ftp_upload/20698/20698_Figure2.jpg" /> 
Figure 2: Density gradient, with cells added-on after centrifugation. The layers are outlined and better visualized on the cartoon depiction. The most superficial layer (0) is generally debris from the spinal cord tissue. Layer 1 is rich in oligodendrocytes and astrocytes. Layers 2 and 3 contain the majority of neurons. While layer 3 contains neurons with the highest purity, some supporting cells (i.e., astrocytes and oligodendrocytes) are found in layer 2. The pellet at the bottom mainly contains microglial cells.

[INSERT FIGURE1 HERE]

Figure 1: Spinal column dissected and spinal cord released from a 3 day-old neonatal mouse. The arrow in (a) points towards the caudal end of the spine, where a needle is inserted to release the spinal cord. A released spinal cord (b) is shown submerged in PBS.

[INSERT FIGURE2 HERE]

Figure 2: Density gradient, with cells added-on after centrifugation. The layers are outlined and better visualized on the cartoon depiction. The most superficial layer (0) is generally debris from the spinal cord tissue. Layer 1 is rich in oligodendrocytes and astrocytes. Layers 2 and 3 contain the majority of neurons. While layer 3 contains neurons with the highest purity, some supporting cells (i.e., astrocytes and oligodendrocytes) are found in layer 2. The pellet at the bottom mainly contains microglial cells.

Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies

Source: Eldeiry, M. et al. Spinal Cord Neurons Isolation and Culture from Neonatal Mice. J. Vis. Exp. (2017)
In this video we describe a step-by-step ...

To isolate spinal cord neurons, take a vertebral column of a neonatal mouse and place it onto a Petri dish. Flush the vertebral column with saline buffer to release the spinal cord. Transfer the spinal cord into cold neuronal medium to promote cell survival.
Next, mince the spinal cord and transfer the fragments into a tube containing fresh neuronal&#160; medium. Incubate the tube in a shaker water bath for acclimatization. After incubation, remove the spent medium and add the digestion medium. Digestive enzymes cleave the tissue matrix to loosen up the cells.
Perform trituration by aspirating the tissue fragments into the pipette and swiftly empty the components, releasing individual cells into suspension. Then, transfer this suspension into a tube containing density gradient medium and centrifuge.
Following centrifugation, the tube&#39;s superficial layer contains tissue debris. The first layer includes oligodendrocytes and astrocytes. The second and third layers comprise neurons, with the third layer having the highest purity. Lastly, the pellet contains microglial cells.
Collect the third layer into a fresh tube and add neuronal medium to dilute out the density gradient medium. Centrifuge to pelletize the neurons. Discard the supernatant and resuspend the pellet in a neurobasal medium to preserve the neurons for further experiments.

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1.4K Views. Source: Eldeiry, M. et al. Spinal Cord Neurons Isolation and Culture from Neonatal Mice. J. Vis. Exp. (2017) In this video we describe a step-by-step protocol to isolate spinal cord neurons from a neonatal mouse for in vitro studies. 

Video: Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies - Concept

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The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

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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.

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Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

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Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies

Transcript

Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

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

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents