Name: generation of oligodendrocytes and oligodendrocyte-conditioned medium for co-culture experiments
Uploaded: 2020-02-09T14:00:00
Description: Watch this Scientific Journal Video about Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments at JoVE.com

The authors would like to thank R&#233;mi Ronzano for his wise advice in manuscript editing. This work was funded by ICM, INSERM, ARSEP foundation grant to NSF, and Bouvet-Labruy&#232;re price.

The care and use of rats in this experiment conforms to institutional policies and guidelines (UPMC, INSERM, and European Community Council Directive 86/609/EEC). The following protocol is established for a standard litter of 12 pups.
1. Preparation of the flasks (~5 min)
NOTE: Perform the following steps the day before dissection in a laminar flow hood under sterile conditions.
<ol>
	<li>Coat the 150 cm2 flasks (T150) with filter cap (1 flask for 2 pup...

<ol>
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Here, we provide a detailed protocol to obtain highly enriched oligodendroglial lineage cell cultures from mixed glial cultures, adapted from a previously published method16, and the subsequent production of OL-conditioned medium. This shaking technique is not expensive, can be repeated three times and is optimal to obtain high quantity of purified OLs, as cells cultured in Bottenstein-Sato (BS) medium containing PDGF&#945; proliferate. Glial cells are prepared using cerebral cortices of Wistar ra...

Oligodendrocytes (OLs) are glial cells of the central nervous system (CNS) that generate myelin wrapping around axons. OLs originate from oligodendrocyte precursor cells (OPCs) which proliferate within the ventricular zones of the embryonic CNS and then migrate and differentiate into fully mature OLs (i.e., myelin-forming cells)1. OPCs are abundant during early development, but also persist in the adult brain where they represent the major proliferative cell population2. A single OL ensheathes multiple axons in non-excitable sections (i.e., internodes), and the edge of each myelin loop attaches to the axon forming the paranodal domain which is crucial for the insulating properties of myelin1,3. In between the paranodes are small unmyelinated gaps called the nodes of Ranvier. These nodes are rich in voltage-gated sodium channels (Nav), allowing the regeneration and rapid propagation of action potentials through saltatory conduction4. This tight interaction also enables axonal energy support through neuronal uptake of lactate from OLs5,6.
The maturation of oligodendroglial lineage cells and the myelination process are tightly regulated by their interactions with neurons7. Indeed, OLs and OPCs, also named NG2 cells, express an array of receptors for neurotransmitters, and can receive input from excitatory and inhibitory neurons, allowing them to sense neuronal activity that can trigger their proliferation and/or differentiation into myelinating cells2. In turn, OPCs/OLs secrete microvesicles and proteins into the extracellular space which alone or synergistically mediate neuromodulative and neuroprotective functions8,9,10,11,12. However, the molecular mechanisms controlling the multiple modes of interactions between oligodendroglial lineage cells and neurons are yet to be fully deciphered.
Moreover, in several CNS pathological conditions, OLs are primarily affected, thus disturbing their interaction with neurons. For instance, in Multiple Sclerosis (MS), neurological dysfunction is caused by focal demyelination in the CNS, secondary to OLs loss that can lead to axonal damage and related disability accumulation. Remyelination can take place, albeit insufficiently in most cases13. Progress in the last decade, due to the development of immunotherapies, have reduce the relapse rate but promoting remyelination remains to date an unmet need. As such, a better understanding of OLs role, functions and influences is of particular interest to the development of new therapies for a wide spectrum of CNS conditions.
Here, we describe the methods of OLs purification and culture. This enables precise examination of intrinsic mechanisms regulating their development and biology. In addition, such highly enriched OLs cultures allow the production of oligodendrocyte-conditioned medium (OCM), which can be added to purified neuron cultures to gain insight into the impact of OLs-secreted factors on neuronal physiology and connectivity. Furthermore, we describe how to implement an in vitro co-culture system where purified oligodendrocytes and neurons are combined together, allowing to address the mechanisms regulating (re)myelination.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-fluorodeoxyuridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase (Deoxyribonuclease I)</td>
			<td>Worthington</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>ThermoFisher</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Sigma</td>
			<td>32221-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>VWR Chemicals</td>
			<td>83801.360</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>ThermoFisher</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>ThermoFisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline without calcium and magnesium</td>
			<td>ThermoFisher</td>
			<td>A1285601</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine(PEI)</td>
			<td>Sigma</td>
			<td>P3143</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetraborate decahydrate</td>
			<td>Sigma</td>
			<td>B9876</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottenstein-Sato (BS) media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>apo-Transferrin human</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (Bovine Serum Albumin)</td>
			<td>Sigma</td>
			<td>A4161</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>ThermoFisher</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I5500</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF</td>
			<td>Peprotech</td>
			<td>AF-100-13A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 (3,3&#39;,5-Triiodo-L-thyronine sodium salt)</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 (L-Thyroxine)</td>
			<td>Sigma</td>
			<td>T1775</td>
			<td></td>
		</tr>
		<tr>
			<td>Co-culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>apo-Transferrin human</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (Bovine Serum Albumin)</td>
			<td>Sigma</td>
			<td>A4161</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceruloplasmin</td>
			<td>Sigma</td>
			<td>239799</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>ThermoFisher</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I5500</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>ThermoFisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescin</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human CNTF</td>
			<td>Sigma</td>
			<td>450-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 (3,3&#39;,5-Triiodo-L-thyronine sodium salt)</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin B12</td>
			<td>Sigma</td>
			<td>V6629</td>
			<td></td>
		</tr>
		<tr>
			<td>Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Sartorius</td>
			<td>514-7010</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Terumo</td>
			<td>1611127</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Dutscher</td>
			<td>193100</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Corning Life Science</td>
			<td>734-1867</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Corning Life Science</td>
			<td>734-1869</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Dutscher</td>
			<td>067003</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-823</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11152-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14008-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Swann-morton</td>
			<td>233-5528</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Infors HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical spoon</td>
			<td>Bar Naor Ltd</td>
			<td>BN2706</td>
			<td></td>
		</tr>
		<tr>
			<td>T150 cm2 flask with filter cap</td>
			<td>Dutscher</td>
			<td>190151</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P2 Wistar rat</td>
			<td>Janvier</td>
			<td>RjHAn:WI</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this protocol, OL lineage cells are purified from glial cultures by shaking off astrocytes and microglia. Purity and phenotypic examination of OL cultures can be assessed by immunostaining with glial markers15. Analysis of the expression of different markers indicated that OL cultures were mostly pre-OLs with 90% &#177; 4% of O4+ cells, 85% &#177; 7% NG2+ cells, and 4.7% &#177; 2.1% of PLP+ cells, while 7.2% &#177; 2.5% of cells ...

Watch this Scientific Journal Video about Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments at JoVE.com

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

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.

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

Research

JoVE Journal

Neuroscience

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding ...

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...