Name: preparation of primary mixed glial cultures from adult mouse spinal cord tissue
Uploaded: 2016-11-19T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue at JoVE.com

Adult spinal cord mixed cultures were first developed using rats with grant support from an NIH/NIDA R01 award (PI De Leo) and an NIH T32 training grant (PI Green). These methods were further adapted for mouse spinal cords with support from the following grants: NIH/NIDA 5K01DA023503 (PI Cao), NIH/NINDS 5R21NS066130 (PI Cao), and NIH/NIGMS P20GM103643 (PI Meng). The authors would also like to thank Dr. Alejandro M. S. Mayer, Department of Pharmacology, Chicago College of Osteopathic Medicine, for his technical help in obtaining microglia-rich mixed glial cells.

The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of New England. The following protocol is for preparing glial cultures from 4 adult mouse spinal cords.
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,...

<ol>
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It is critical to perform all steps-including the media changing schedule-in a consistent manner in order to obtain reproducible results between experiments. The following steps are particularly critical in obtaining excellent quality adult mixed glia.
The enzymatic digestion is an important aspect of this protocol. It is crucial that the tissue pieces are well-digested in order to obtain a single cell suspension. However, over-digestion will result in significantly fewer cells. Each lab needs...

Chronic pain is a serious health problem that affects approximately 100 million adults in the United States, with an estimated annual cost of up to $635 billion1. Mounting evidence has indicated a significant contribution of spinal cord glial cells in the development of neuropathic pain, one of the most devastating kinds of chronic pain2. A proper in vitro culture system would help significantly in further investigating the roles of glial cells at the cellular and molecular levels.
Currently, the in vitro glial culture systems utilized by researchers include mainly glial cells derived from cortical tissues of neonatal mouse or rat brains and immortalized glial cell lines derived from neonatal mouse or rat primary glial cells. Neonatal cells contain significant numbers of undifferentiated cells that can be further differentiated into glial cells (mainly astrocytes and microglia), thus providing abundant cells for experimental usage3. However, our previous study has shown that neonatal brain glial cells responded significantly differently from adult spinal cord glial cells upon lipopolysaccharide (LPS) stimulation. For example, interleukin (IL)-4 displayed enhanced inhibitory effects on LPS-induced nitric oxide (NO) production in adult rat spinal cord glia compared to neonatal brain glia4. Furthermore, the profiles of chemokine production upon stimulation by a neuropeptide, calcitonin gene-related peptide (CGRP), are unarguably different between neonatal mouse brain glia (methods described in Ref. 5) and adult mouse spinal cord glia (Figure 1). Established cell lines are easy to use and maintain and can provide a large number of cells in a short time period. In general, immortalized cell lines are generated either using a virus-mediated immortalization system (such as the widely-used microglial cell line BV2)6, 7 or following the identification of spontaneous transformation (such as the astrocyte cell line C8-D1A and microglial cell line C8-B4)8, 9. Cell lines are excellent in studying the molecular characteristics of astrocytes and microglia individually; however, the results obtained from cell lines always require further validation in primary cells or in&#160;in vivo conditions. It should also be noted that there has not been a report of a glial cell line that is derived from a rodent spinal cord glia.
To help&#160;investigating&#160;the role of spinal cord glial cells in the development of neuropathic pain, a culture system that is derived from the adult mouse spinal cord has been developed by adapting a previously-reported method used to generate adult rat mixed glial cultures4, 5. The mouse spinal cord glial culture was further refined recently10 and is described in more detail in this article. Adult spinal cord mixed glial cultures can be established with a mouse from selected strains that fit the needs of the particular study, and cells can be maintained in culture for up to 21 d post initiation of the culture. This method can be used in neuropathic pain studies, as well as in investigations of various neurological diseases that involve pathological changes within the spinal cord, such as amyotrophic lateral sclerosis (ALS), human immunodeficiency virus (HIV)-associated sensory neuropathy, and multiple sclerosis (MS).

<table border="0" cellpadding="0" cellspacing="0" width="2053">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Dulbecco&#8217;s modification of Eagle&#8217;s media (DMEM) with 4.5g/L glucose</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:119px;">12-709F</td>
			<td style="width:1168px;">The cDMEM media is a standard, widely used culture media. Individual researcher can decide where to purchase the DMEM and all other component used to make cDMEM media.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">L-Glutamine (100x)</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:119px;">17-605E</td>
			<td>The L-glutamine is a standard, widely used component for various culture media. Individual researcher can decide where to purchase L-glutamine.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Antibiotic-Antimycotic Solution (100x)</td>
			<td style="width:277px;">Corning-Mediatech</td>
			<td style="width:119px;">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 researcher can decide where to purchase individual component.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">2-mercaptoethanol (2-ME)</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td>A BioReagent, suitable for cell culture, molecular biology and electrophoresis.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Papain dissociation system</td>
			<td style="width:277px;">Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>Individual components of this kit can be purchased separately.&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Percoll</td>
			<td>GE Health Care</td>
			<td style="width:119px;">17-0891-01</td>
			<td>Percoll is sold as sterile solution. Undiluted Percoll can be re-autoclaved if needed.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Lab-line incubator/shaker&#160;</td>
			<td style="width:277px;">Barstead/Lab-line</td>
			<td style="width:119px;">MaxQ4000&#160;</td>
			<td>This is the incubator/shaker we have currently. Other types of shakers can be used instead.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Lipopolysaccharides, Salmonella Minnesota Re595&#160;</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:119px;">L-9764</td>
			<td>Other strains of LPS can also induce glial responses. The magnitude of the responses may vary.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse IL-6 DuoSet ELISA&#160;</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY406</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse TNF-alpha DuoSet ELISA</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY410</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse IP-10 (CXCL10) DuoSet ELISA</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY466</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse MCP-1 (CCL2) ELISA Opteia set</td>
			<td style="width:277px;">BD Biosciences</td>
			<td style="width:119px;">555260</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse chemokine Q-Plex</td>
			<td style="width:277px;">Quansys Biosciences</td>
			<td style="width:119px;">120251MS</td>
			<td>This product was available for in-house assay at the time. Instead, we sent out samples to the manufacturer for analysis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">APC-anti-mouse-CD45 (clone 30-F11)</td>
			<td style="width:277px;">eBioscience</td>
			<td style="width:119px;">17-0451</td>
			<td>Antibody of the same clone but from other vendors can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">FITC-anti-mouse-CD11b (clone M1/70)</td>
			<td style="width:277px;">eBioscience</td>
			<td style="width:119px;">11-0112</td>
			<td>Antibody of the same clone but from other vendors can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Accuri C6 flow cytometer</td>
			<td style="width:277px;">BD Biosciences</td>
			<td style="width:119px;">BD Accuri C6&#160;</td>
			<td>Each individual Researcher can use any flow cytometer he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">FlowJo software</td>
			<td style="width:277px;">Tree Star, Inc.</td>
			<td style="width:119px;">FlowJo7.6.5</td>
			<td>Each individual Researcher can use any analysis software he or she prefers.</td>
		</tr>
	</tbody>
</table>

preparation of primary mixed glial cultures from adult mouse spinal cord tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

This method can be used to prepare mixed glial cells from both mice and rats. The average total cell number per well in a 12-well plate on D 12 post-initiation of the culture should be relatively stable, with around 100,000 cells per well when cells are derived from mouse spinal cords. Glial cells obtained from this method can be used in experiments that are designed to examine adult spinal cord glial responses upon administration of substances and agents of interest. Figure 3&#16...

Watch this Scientific Journal Video about Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue at JoVE.com

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

The overall goal of this protocol is to establish primary mixed glial cultures from adult mouse spinal cords for in vitro studies. This method provides us an in vitro system to investigate the roles of glial cells in neurological diseases that involve pathological changes within the spinal cord, such as neuropathic pain and multiple sclerosis. The main advantage of this technique is that the glial cultures are prepared from adult mouse spinal cord, providing a system that is more accurately reflecting the in vivo conditions.Demonstrating the procedure will be Jennifer Malon, a technician from my laboratory. 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 millimeter 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 five milliliter pipet to promote further dissociation. Then, transfer the cell suspension into a 15 milliliter tube and centrifuge for 300 x g for five minutes at room temperature. During the centrifugation, at 300 microliters of reconstituted albumin ovomucoid inhibitor solution to 2.7 milliliters of ABSS in a sterile tube and mix well.Then add 150 microliters of DNAse solution. Following the centifugation, 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 three milliliters of reconstituted albumin ovomucoid inhibitor solution, without DNAse, to the cell suspension. Centrifuge the cells at 70 x g for six 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 eight milliliters of room temperature 20%gradient density medium into the tube containing the cell pellet and vortex gently. Next, centrifuge the cells at 800 x 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 eight milliliters of cDMEM diluted with HBSS. Centrifuge the cells at 400g for 10 minutes at four 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 one 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 until the cells are ready for treatment between days 12 and 14.Mixed glial cells from adult C57 black six mice were cultured at either 37 degrees Celsius or 35.9 degrees Celsius and analyzed via flow cytometry. As you can see, there is no obvious difference in the total cell populations at these temperatures. These representative plots show the CD45 positive CD11b positive microglia populations isolated from the total cell populations previously shown.This figure demonstrates that a higher microglial content can be obtained when mixed glia are cultured at 35.9 degrees Celsius, instead of 37 degrees Celsius. Adult spinal cord, mixed glial cultures were prepared from C67 black six mice. Representative images of the cultured cells at days one, four, eight, and 12 are shown.Images showing the typical progress of the culture, as well as demonstrating the importance of media, change at day one post-culture establishment. Once mastered, the initial set up of mixed glia cell culture can be completed in about four hours, if performed properly. Following the establishment of mixed glia culture, microglia depleted, and microglia enriched cultures, can be obtained from this initial mixed population.However, the yield of microglia enriched cells will be limited unless large number of spinal cords are used to establish cultures. This technique was initially designed to investigate the roles of glial cells during the development of neuropathic pain. However, it can be used to study other neurological diseases that involve pathological changes within the adult spinal cord.

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