isolating and culturing oligodendrocyte precursor cells from mouse pup brain cortex

Isolating and Culturing Oligodendrocyte Precursor Cells from Mouse Pup Brain Cortex

Add 1 milliliter of 0.05% trypsin with 0.53 millimolar EDTA to each tube with tissue. Triturate the mixture with a 10-milliliter pipette approximately 20 times. Then, transfer the cell suspension to an empty 50-milliliter conical tube.
Incubate the solution at 37 degrees Celsius and 5% carbon dioxide for 15 minutes, gently agitating the lysates after eight minutes. After the incubation, add 5 milliliters of MGM, and 200 microliters of DNase-I to each tube for a final concentration of 50 micrograms per milliliter. Triturate each lysate 10 times with a 10-milliliter pipette.
Then, let the cell suspension sit for three minutes at room temperature to allow nondissociated tissue to settle at the bottom of the tube. Transfer the cell suspensions to new 50-milliliter conical tubes, leaving behind the non dissociated tissue. After centrifugation, resuspend the cell pellet in 5 milliliters of MGM and triturate it 20 times.
Plate the cell suspensions on coated T-25 flasks, and incubate the cells at 37 degrees Celsius and 5% carbon dioxide. To perform oligodendrocyte precursor cell isolation, shake the cells for 15 hours. Then, remove the supernatant from the flask and plate it on a sterile Petri dish.
Incubate the supernatant at 37 degrees Celsius and 5% carbon dioxide for 30 minutes, swirling after 15 minutes to remove remaining microglia. Remove non-adherent cell supernatant. Count the cells and plate them on a poly-D-lysine-coated surface at the desired concentration.
Return the cells to the incubator for at least one hour. Then, gently aspirate 95% of media and slowly add warm OPC media, pipetting it against the wall of the well to minimize disruption of OPCs.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Tissue dissociation
NOTE: All the following procedures are carried out in a sterile tissue culture designated biosafety cabinet using aseptic technique and sterile materials.
<ol>
	<li>Add 1 mL of 0.05% trypsin containing 0.53 mM EDTA to each 15 mL conical tube of 9 mL DMEM and tissue to begin tissue lysis. 
	NOTE: DMEM contains a high amount of calcium chloride, which can act as an inhibitor of trypsin. If dissociation is not complete following the outlined steps, Hanks&#8217; Balanced Salt Solution without calcium or magnesium can be used. After trypsinization, the enzyme can be neutralized by adding a trypsin inhibitor, although calcium should be added back to the solution as it is a cofactor for DNase I, which is used in subsequent steps.</li>
	<li>Triturate with a 10 mL pipette approximately 20x.</li>
	<li>Transfer the cell suspensions to empty 50 mL conical tubes.</li>
	<li>Incubate the solution at 37 &#176;C, 5% CO2 for 15 min, gently agitating the lysates after 8 min.</li>
	<li>Add 5 mL of MGM and 200 &#181;L (5 mg/mL) DNase I to each tube for a final concentration of 50 &#181;g/mL.</li>
	<li>Triturate each lysate with a 10 mL pipette 10x.</li>
	<li>Let the cell suspensions sit for 3 min at room temperature to allow non-dissociated tissue to settle at the bottom of the tubes.</li>
	<li>Transfer the cell suspensions to new 50 mL conical tubes, leaving behind the non-dissociated tissue. 
	NOTE: The lysis and trituration steps described above significantly limits the amount of non-dissociated tissue.</li>
	<li>Centrifuge the tubes at 300 x g for 3 min at 4 &#176;C without brake.</li>
	<li>Aspirate the supernatant and resuspend the remaining cell pellets in 5 mL of MGM.</li>
	<li>Triturate the pellet with a 5 mL pipette 20x.</li>
	<li>Plate the 5 mL cell suspensions on coated T25 flasks.</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 and change media initially after 24 h to remove cell debris. 
	NOTE: Some protocols recommend an initial media change after 72 h. Optimization of this step may be required.</li>
	<li>Perform a 100% media change with MGM every 48-72 h until cells are 80% confluent (approximately 5-7 days). 
	NOTE: All media must be warmed to 37 &#176;C before media changes.</li>
</ol>
2. Oligodendrocyte precursor cell isolation
When plating OPCs following initial isolation, they must be plated on a poly-D-lysine-coated surface (sterile plate or coverslip). Prepare these materials before completing this section.
<ol>
	<li>Following 15 h shake, remove the supernatant from the flasks and plate on a sterile 100 mm Petri dish.</li>
	<li>Incubate the supernatant at 37 &#176;C, 5% CO2 for 30 min, swirling after 15 min to remove remaining microglia, as these will very quickly adhere to the dish. Non-tissue culture-treated Petri dishes may be used for this step.</li>
	<li>Remove non-adherent cell supernatant, count, and plate on a poly-D-lysine-coated surface. Typically, 7,500-10,000 OPCs are plated/cm2.</li>
	<li>Incubate at 37 &#176;C, 5% CO2 for at least 1 h (up to 6 h), then gently aspirate 95% of media, and slowly add warm OPC Media, pipetting media against the wall of the well to minimize disruption of OPCs. Change media every 48 h until cells are ready for use. 
	NOTE: It is critical that only one well is changed at a time. OPCs are sensitive and are especially intolerant to dry conditions. The addition of PDGF-AA in OPC media is to delay OPC maturation into oligodendrocytes. This factor may be excluded from culture media if the experimental focus is mature oligodendrocytes.</li>
</ol>
Table 1: Buffer and Media Recipes.
Mixed Glia Media (MGM)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>88 mL</td>
			<td>1X DMEM (high glucose, w/L-glutamine, w/Na pyruvate)</td>
		</tr>
		<tr>
			<td>10 mL</td>
			<td>Heat-inactivated FBS</td>
		</tr>
		<tr>
			<td>1.0 mL</td>
			<td>L-Glutamine (100X)</td>
		</tr>
		<tr>
			<td>1.0 mL</td>
			<td>Antibiotic/Antimycotic</td>
		</tr>
	</tbody>
</table>
OPC Media (50mL)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>49 mL</td>
			<td>Neurobasal media</td>
		</tr>
		<tr>
			<td>1.0 mL</td>
			<td>B27 supplement (50X)</td>
		</tr>
		<tr>
			<td>10 ng/ml&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>PDGF-AA</td>
		</tr>
		<tr>
			<td colspan="2">NOTE: PDGF-AA is added fresh prior to each media change.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin and 0.53 mM EDTA</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td>Tissue dissociation</td>
		</tr>
		<tr>
			<td>1X PBS pH 7.4</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>Standard reagent</td>
		</tr>
		<tr>
			<td>50 mL, 25 cm square cell culture flask</td>
			<td>Greiner Bio-One</td>
			<td>690 175</td>
			<td>Cell culture (T25) flask</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic 100X</td>
			<td>Gibco</td>
			<td>15240-096</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>B-27 Supplement 50X</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9647-50G</td>
			<td>Antibody diluent</td>
		</tr>
		<tr>
			<td>DMEM (1X), high glucose with Na pyruvate</td>
			<td>Gibco</td>
			<td>11995040</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma</td>
			<td>10104159001</td>
			<td>Tissue dissociation</td>
		</tr>
		<tr>
			<td>Fetal bovine serum heat inactivated</td>
			<td>Gibco</td>
			<td>A3840001</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (w/o Ca or Mg)</td>
			<td>ThermoFisher</td>
			<td>14170120</td>
			<td>Tissue dissociation</td>
		</tr>
		<tr>
			<td>L-glutamine, 200mM</td>
			<td>Gibco</td>
			<td>20530081</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td>Blocking solution component</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine 12 mm #1 German Glass Coverslip</td>
			<td>Corning Biocoatt</td>
			<td>354086</td>
			<td>Cell adherent</td>
		</tr>
	</tbody>
</table>

Source: Sinyuk, M., et al. <a href="https://app.jove.com/v/61345">Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions.</a> J. Vis. Exp. (2020)
This video demonstrates a technique for isolating and culturing oligodendrocyte precursor cells (OPCs) from the mouse pup brain cortex.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Tissue dissociation
NOTE: All the ...

Take a mouse pup cortex.
Add a proteolytic enzyme that digests the tissue&#39;s extracellular matrix. Then, mechanically dissociate the tissue, loosening the cells.
Add glial media and DNase to degrade any contaminating DNA.&#160;
Mechanically dissociate the tissue to release neurons and glial cells.&#160;
Once the undigested tissue settles, collect the media containing the cells and centrifuge. Remove the supernatant.
Resuspend cells in glia media and plate them on an extracellular matrix protein-coated flask.&#160;
Incubate. Astrocytes adhere more strongly to the coated surface than other cells. Glial media promote only glial cell survival.&#160;
Replace the media to remove debris.
Incubate with shaking to detach the overlying oligodendrocyte precursor cells, OPCs, and microglia.
Transfer the media with the cells onto a culture plate.
Incubate with shaking to facilitate differential microglial adhesion.
Transfer the non-adherent OPC supernatant onto a poly-D-lysine-coated multi-well plate. Incubate for OPC adherence.
Replace the media with OPC media, allowing OPC growth.

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JoVE Journal

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In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

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The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
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Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
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Isolating and Culturing Oligodendrocyte Precursor Cells from Mouse Pup Brain Cortex

TRANSKRIPT

Isolating and Culturing Oligodendrocyte Precursor Cells from Mouse Pup Brain Cortex