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EoE Sub Articles

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>

establishing primary mixed glial cell cultures from a mouse spinal cord

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.

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

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.

establishing-primary-mixed-glial-cell-cultures-from-mouse-spinal

Research

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Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

71 Views. Source: Malon, J. T., et al. Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue. 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. 

Video: Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord - Concept

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

JoVE Journal

Developmental Biology

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Bioengineering

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

Transcript

Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse