establishing primary mixed glial cell cultures from a mouse spinal cord

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

Working in a tissue culture hood, transfer four mouse spinal cords into a Petri dish of HBSS. Use sterile scissors and forceps to cut each of the spinal cords into small pieces. Then, transfer the pieces to a 50-milliliter conical tube containing papain DNase enzyme mixture. Avoid transferring HBSS to the enzyme mixture as this may result in decreased performance of the enzyme.
It is crucial that tissue pieces are well-digested to obtain a single-cell suspension. However, over-digestion will result in fewer viable cells. Each lab needs to perform pilot tests to determine the exact digestion time based on what works best for their cells.
Next, gently vortex the tube and then incubate at 37 degrees Celsius for one hour with orbital shaking at 150 rotations per minute. Following the incubation, vortex the tube, and then vigorously triturate the tissue using a 5-milliliter pipette to promote further dissociation. Then, transfer the cell suspension into a 15-milliliter tube, and centrifuge at 300 times g for 5 minutes at room temperature.
During the centrifugation, add 300 microliters of reconstituted albumin ovomucoid inhibitor solution to 2.7 milliliters of EBSS in a sterile tube and mix well. Then, add 150 microliters of DNase solution. Following the centrifugation, remove the supernatant and resuspend the cell pellet in the freshly prepared albumin ovomucoid inhibitor and DNase solution. Vortex well to break the cell pellet.
Then, add 3 milliliters of reconstituted albumin ovomucoid inhibitor solution without DNase to the cell suspension. Centrifuge the cells at 70 times g for 6 minutes at room temperature. After the spin, remove the supernatant which contains membrane fragments, and retain the pellet.
To remove myelin from the dissociated spinal cord cells, first, add 8 milliliters of room temperature 20% density gradient medium into the tube containing the cell pellet and vortex gently. Next, centrifuge the cells at 800 times g for 30 minutes at room temperature without breaking. Following centrifugation, carefully aspirate the top layer of debris, which contains mostly myelin, and the supernatant leaving the pellet.
To remove any remaining density gradient, wash the cells by resuspending the pellet with 8 milliliters of cDMEM diluted with HBSS. Centrifuge the cells at 400 g for 10 minutes at 4 degrees Celsius. Remove the supernatant and wash the cells with diluted cDMEM as before.
After removing the supernatant, the pellet can be stored on ice until seeding the cells. Once ready for plating, resuspend the cells in 14 milliliters of cDMEM supplemented with 2-Mercaptoethanol, and add 1 milliliter of the cell suspension to each well in a 12-well plate.
Plate extra wells that can be used to determine the average cell number per well, and the microglial content of the culture. Once the cells have been plated, incubate the cells at 35.9 degrees Celsius with 5% carbon dioxide. Change the medium on day 1, which is the day after plating.
Some cells are attached to the culture plate, but they are still mostly round. There are also many floating cells and significant debris. Repeat the media change three to four days thereafter.

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1. Preparation of Solutions in a Culture Hood under Aseptic Conditions
<ol>
	<li>Prepare culture media: complete Dulbecco&#39;s modification of Eagle&#39;s media (cDMEM) containing DMEM (with 4.5 g/L glucose), 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 250 ng/mL Amphotericin B, and 50 &#181;M 2-mercaptoethanol (2-ME; see 1.1.1). Mix and filter sterilize all other components and then add FBS. Store the culture media at 4 &#176;C.
	<ol>
		<li>Prepare 50 mM 2-ME/phosphate-buffered saline (PBS) stock solution: add 100 &#181;L of concentrated 2-ME (14.3 M) into 28.6 mL of 1x PBS, and filter sterilize (the solution can be stored at 4 &#176;C for several months). Use 1 mL of 2-ME stock solution for every 1,000 mL of culture media.</li>
	</ol>
	</li>
	<li>Preparation of the density gradient media
	<ol>
		<li>Prepare a stock isotonic density gradient media solution (e.g., 100% Percoll) from 1 part 10x regular sterile PBS and 9 parts sterile stock density gradient media (e.g., Percoll).</li>
		<li>Dilute the 100% density gradient media (made in 1.2.1) with regular 1x sterile PBS to make a 20% density gradient media (e.g., 10 mL of 100% density gradient media with 40 mL of 1x PBS) and store it at 4 &#176;C. Bring the 20% density gradient media to room temperature (RT) before preparing the cell culture.</li>
	</ol>
	</li>
	<li>Preparation of solutions from the papain dissociation system 
	&#8203;NOTE: The papain dissociation system can be identified in the Materials Table. Other similar dissociation kits (including those assembled in-house) can also be used. All components must be sterile.
	<ol>
		<li>Add 32 mL of Earle&#39;s Balanced Salt Solution (EBSS) to the ovomucoid inhibitor mixture (powder).</li>
		<li>Add 5 mL of EBSS to one papain vial (powder). Place the papain vial in a 37 &#176;C water bath for 10 min or until the papain is dissolved.</li>
		<li>Add 500 &#181;L of EBSS to one DNase vial (powder). Mix gently.</li>
		<li>Add 250 &#181;L of the DNase solution (above) to the papain vial.</li>
		<li>Calculate the total amount of the above papain/DNase mixture needed (approximately 800 &#181;L of papain/DNase mixture per mouse spinal cord) and transfer the needed mixture into a 50 mL tube for tissue digestion. Store the leftover in the original vial at 4 &#176;C for at least two weeks.</li>
		<li>Keep all components from the kits on ice until needed.</li>
	</ol>
	</li>
	<li>Prepare spinal cord collection tubes: one sterile 15 mL tube with 5 mL of Hank&#39;s balanced Salt Solution (HBSS; from the papain dissociation system, for collecting spinal cords) and one sterile 15 mL tube with 1x PBS (for filling up the 5 mL syringe). In addition, prepare one sterile petri dish (35 mm or 60 mm) with 5 mL of HBSS and keep it in the culture hood.</li>
</ol>
2. Preparation of the Single Cell Suspension
Note: Perform steps 3 and 4 in a culture hood to keep everything sterile.
<ol>
	<li>Transfer all spinal cords into the HBSS-containing petri dish. Cut each of the spinal cords into many fine, small pieces with sterile scissors and forceps. Transfer the spinal cord tissue to the 50-mL conical tube containing the prepared papain/DNase enzyme mixture (step 1.3.5) using sterile forceps or a 10 mL pipette (avoid adding HBSS to the enzyme mixture, as this will further dilute the prepared enzyme solution and may result in decreased performance of the enzyme).</li>
	<li>Vortex the tube gently to mix. Incubate the tube at 37 oC for 1 h in an incubator/shaker with orbital shaking at 150 rpm.</li>
	<li>Vortex the tube again and vigorously triturate the enzyme solution with the tissue using a 5 mL pipette to promote further dissociation.</li>
	<li>Transfer the cell suspension into a 15-mL tube and centrifuge at 300 x g for 5 min at RT.</li>
	<li>During centrifugation, mix 2.7 mL EBSS with 300 &#181;L of reconstituted albumin-ovomucoid inhibitor solution (1.3.1) in a sterile tube. Add 150 &#181;L of the DNase solution (step 1.3.3).</li>
	<li>Following centrifugation, remove the supernatant and resuspend the cell pellet with the solution prepared above (step 1.5). Vortex well to break the cell pellet.</li>
	<li>Add 3 mL of reconstituted albumin-ovomucoid inhibitor solution (step 1.4.1) to the cell suspension. Centrifuge cells at 70 x g for 6 min at RT. Remove the supernatant (which contains membrane fragments).</li>
</ol>
3. Further Removal of Myelin from the Single Cell Suspension
<ol>
	<li>Add 8 mL of 20% density gradient media (prepared in step 1.2.2) into the tube containing the cell pellet, vortex gently to disrupt the pellet, and centrifuge the cells at 800 x g for 30 min at RT without braking. Carefully remove the top layer of debris (mostly myelin) and the supernatant but keep the pellet.</li>
	<li>To remove remnants of the density gradient, wash the cells by resuspending the cell pellet with 8 mL of a diluted cDMEM (1 part cDMEM and 2 parts HBSS). Centrifuge the cells at 400 x g for 10 min at 4 oC. Remove the supernatant and wash the cells again with the diluted cDMEM (above) in the same manner. Keep the cells on ice until seeding them.</li>
	<li>Remove the supernatant and resuspend the cell pellet in culture media (cDMEM supplied with 2-ME (prepared in step 1.1)). For one 12-well plate, use 3 mL x 4 (number of mice used) + 2 mL = 14 mL media. This will ensure that there is sufficient cell suspension for the entire plate (12 wells) and will provide for extra wells that can be used to determine the average cell number per well and the microglial content of the culture. If other types of culture vessels will be used, calculate the total volume of needed culture media proportionally.</li>
	<li>Add 1 mL of the cell suspension into each well of a 12-well plate.</li>
	<li>Incubate the cells at 35.9 oC with 5% CO2.</li>
	<li>Change the media (remove old media via aspiration) on D 1 and then every 3 - 4 d thereafter (typically change the media on D: 1, 4, 8, and 11, and then use the cells on day 12). 
	NOTE: On day 1, the culture may contain significant amounts of debris due to the residue myelin from the spinal cord tissue; thus, changing the media on day 1 is recommended. Cultures are ready for treatment between D 12 - 14. Cells usually are 80% confluent at D 12 and can be near 100% confluent by D 14. Typically, on D 12, there are about 100,000 cells per well in a 12-well plate.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s modification of Eagle&#39;s media (DMEM) with 4.5 g/L glucose</td>
			<td>Lonza</td>
			<td>12-709F</td>
			<td>The cDMEM media is a standard, widely used culture media. 
			&#160;Individual researchers can decide where to purchase the DMEM and all other components used to make cDMEM media.</td>
		</tr>
		<tr>
			<td>L-Glutamine (100x)</td>
			<td>Lonza</td>
			<td>17-605E</td>
			<td>The L-glutamine is a standard, widely used component for various culture media. Individual researchers can decide where to purchase L-glutamine.</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution (100x)</td>
			<td>Corning-Mediatech</td>
			<td>30-004-CI</td>
			<td>This is a combination of penicillin, streptomycin and Amphotericin formulated to contain 10,000 units/mL penicillin G, 10 mg/mL streptomycin sulfate and 25 &#181;g/ mL amphotericin B. Individual researchers can decide where to purchase individual components.</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol (2-ME)</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td>A BioReagent, suitable for cell culture, molecular biology and electrophoresis.</td>
		</tr>
		<tr>
			<td>Papain dissociation system</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>Individual components of this kit can be purchased separately.</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Health Care</td>
			<td>17-0891-01</td>
			<td>Percoll is sold as sterile solution. Undiluted Percoll can be re- autoclaved if needed.</td>
		</tr>
		<tr>
			<td>Lab-line incubator/shaker</td>
			<td>Barstead/Lab-line</td>
			<td>MaxQ4000</td>
			<td>This is the incubator/shaker we have currently. Other types of shakers can be used instead.</td>
		</tr>
	</tbody>
</table>

Source: Malon, J. T., et al. <a href="https://app.jove.com/v/54801">Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for isolating and culturing primary mixed glial cells from adult mouse spinal cord tissue. The protocol involves enzymatic digestion of the tissue, followed by the removal of broken cell membranes and myelin debris to obtain a single-cell population. The cells are then cultured in media optimized for mixed glial cell growth.

1. Preparation of Solutions in a Culture Hood under Aseptic Conditions

	Prepare culture media: complete Dulbecco&#39;s modification of Eagle&#39;s media ...

Harvest mouse spinal cords and cut them into fragments.
Transfer the fragments to a tube containing a proteolytic enzyme and DNase. Vortex and incubate.
The enzyme digests the tissue&#39;s extracellular matrix, while DNase degrades contaminating free DNA.
Vortex again and pipette repeatedly to complete tissue dissociation, obtaining a single-cell suspension.
Centrifuge and remove the supernatant. Resuspend the cells in a solution containing DNase, and a protease inhibitor, and vortex. The inhibitor neutralizes the enzyme.
Layer fresh inhibitor solution on the top, centrifuge, and remove the supernatant.
Resuspend the cells in density gradient media, vortex, and then centrifuge.
The cells settle at the bottom, while the myelin debris remains at the top.
Discard the debris. Wash repeatedly with buffer, centrifuge, and remove the supernatant. Resuspend the cells in growth media.
Plate the cells. The uncoated surface facilitates glial cell attachment.
Remove the spent media and add fresh media to induce glial cell proliferation, establishing a mixed glial culture.

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Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

Transcripción

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord