Name: Video: عزل واستزراع الخلايا السليفة قليلة التغصن من قشرة دماغ جرو الفأر - Concept
Uploaded: 2024-08-30T13:23:57.000+00:00
Duration: 1 min 33 s
Description: 149 Views. المصدر: Sinyuk, M., et al. تشريح وعزل الخلايا الدبقية الفئرانية من مناطق الجهاز العصبي المركزي المتعددة. J. Vis. Exp. (2020) يوضح هذا الفيديو تقنية لعزل واستزراع الخلايا السليفة قليلة التغصن (OPCs) من قشرة دماغ جرو الفأر.

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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>

isolating and culturing oligodendrocyte precursor cells from mouse pup brain cortex

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.

Isolating and Culturing Oligodendrocyte Precursor Cells from 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.

isolating-culturing-oligodendrocyte-precursor-cells-from-mouse-pup

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

149 Views. المصدر: Sinyuk, M., et al. تشريح وعزل الخلايا الدبقية الفئرانية من مناطق الجهاز العصبي المركزي المتعددة. J. Vis. Exp. (2020) يوضح هذا الفيديو تقنية لعزل واستزراع الخلايا السليفة قليلة التغصن (OPCs) من قشرة دماغ جرو الفأر. 

Video: عزل واستزراع الخلايا السليفة قليلة التغصن من قشرة دماغ جرو الفأر - Concept

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

إنتاج واستخدام الفيروسة البطيئة إلى خلايا انتقائي تنبيغ الابتدائية دبقية قليلة التغصن السلائف ل<em&gt; في المختبر</em&gt; فحوصات تكون الميالين

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

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Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

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.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

جيل من Oligodendrocytes وOligodendrocyte- متوسطة مكيفة للتجارب الثقافة المشتركة

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

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.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
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.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

عالية المحتوى فحص التمايز وتحليل النضج من الجنين والكبار الخلايا الجذعية العصبية المشتقة من ثقافات الخلايا السليفة Oligodendrocyte

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

Isolating and Culturing Oligodendrocyte Precursor Cells from Mouse Pup Brain Cortex

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Isolating and Culturing Oligodendrocyte Precursor Cells from Mouse Pup Brain Cortex