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>
	<li>Zalc, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acquisition+of+myelin:+a+success+story.">The acquisition of myelin: a success story.</a> Novartis Foundation Symposium. 276, 15-21 (2006).</li><li>Habermacher, C., Angulo, M. C., Benamer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+versus+GABA+in+neuron-oligodendroglia+communication.">Glutamate versus GABA in neuron-oligodendroglia communication.</a> Glia. 67 (11), 2092-2106 (2019).</li><li>Sherman, D. L., Brophy, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+axon+ensheathment+and+myelin+growth.">Mechanisms of axon ensheathment and myelin growth.</a> Nature Reviews. Neuroscience. 6 (9), 683-690 (2005).</li><li>Freeman, S. A., Desmazi&#232;res, A., Fricker, D., Lubetzki, C., Sol-Foulon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sodium+channel+clustering+and+its+influence+on+axonal+impulse+conduction.">Mechanisms of sodium channel clustering and its influence on axonal impulse conduction.</a> Cellular and molecular life sciences: CMLS. 73 (4), 723-735 (2016).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroglia+metabolically+support+axons+and+contribute+to+neurodegeneration.">Oligodendroglia metabolically support axons and contribute to neurodegeneration.</a> Nature. 487 (7408), 443-448 (2012).</li><li>Nave, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+and+the+trophic+support+of+long+axons.">Myelination and the trophic support of long axons.</a> Nature Reviews. Neuroscience. 11 (4), 275-283 (2010).</li><li>Monje, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+Plasticity+and+Nervous+System+Function.">Myelin Plasticity and Nervous System Function.</a> Annual Review of Neuroscience. 41, 61-76 (2018).</li><li>Birey, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+Stress-Induced+Loss+of+NG2+Glia+Triggers+Emergence+of+Depressive-like+Behaviors+through+Reduced+Secretion+of+FGF2.">Genetic and Stress-Induced Loss of NG2 Glia Triggers Emergence of Depressive-like Behaviors through Reduced Secretion of FGF2.</a> Neuron. 88 (5), 941-956 (2015).</li><li>Fr&#252;hbeis, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter-triggered+transfer+of+exosomes+mediates+oligodendrocyte-neuron+communication.">Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication.</a> PLoS Biology. 11 (7), e1001604 (2013).</li><li>Jang, M., Gould, E., Xu, J., Kim, E. J., Kim, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocytes+regulate+presynaptic+properties+and+neurotransmission+through+BDNF+signaling+in+the+mouse+brainstem.">Oligodendrocytes regulate presynaptic properties and neurotransmission through BDNF signaling in the mouse brainstem.</a> eLife. 8, (2019).</li><li>Sakry, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+precursor+cells+modulate+the+neuronal+network+by+activity-dependent+ectodomain+cleavage+of+glial+NG2.">Oligodendrocyte precursor cells modulate the neuronal network by activity-dependent ectodomain cleavage of glial NG2.</a> PLoS Biology. 12 (11), e1001993 (2014).</li><li>Sakry, D., Yigit, H., Dimou, L., Trotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+precursor+cells+synthesize+neuromodulatory+factors.">Oligodendrocyte precursor cells synthesize neuromodulatory factors.</a> PloS One. 10 (5), e0127222 (2015).</li><li>Stadelmann, C., Timmler, S., Barrantes-Freer, A., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+in+the+Central+Nervous+System:+Structure,+Function,+and+Pathology.">Myelin in the Central Nervous System: Structure, Function, and Pathology.</a> Physiological Reviews. 99 (3), 1381-1431 (2019).</li><li>Freeman, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+conduction+velocity+linked+to+clustering+of+nodal+components+precedes+myelination.">Acceleration of conduction velocity linked to clustering of nodal components precedes myelination.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (3), E321-E328 (2015).</li><li>Baumann, N., Pham-Dinh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+oligodendrocyte+and+myelin+in+the+mammalian+central+nervous+system.">Biology of oligodendrocyte and myelin in the mammalian central nervous system.</a> Physiological Reviews. 81 (2), 871-927 (2001).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> The Journal of Cell Biology. 85 (3), 890-902 (1980).</li><li>Dean, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-specific+differences+in+perinatal+rodent+oligodendrocyte+lineage+progression+and+its+correlation+with+human.">Strain-specific differences in perinatal rodent oligodendrocyte lineage progression and its correlation with human.</a> Developmental Neuroscience. 33 (3-4), 251-260 (2011).</li><li>Domingues, H. S., Portugal, C. C., Socodato, R., Relvas, J. B., Astrocyte, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte,+Astrocyte,+and+Microglia+Crosstalk+in+Myelin+Development,+Damage,+and+Repair.">Oligodendrocyte, Astrocyte, and Microglia Crosstalk in Myelin Development, Damage, and Repair.</a> Frontiers in Cell and Developmental Biology. 4, 71 (2016).</li><li>Klinghoffer, R. A., Hamilton, T. G., Hoch, R., Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+allelic+series+at+the+PDGFalphaR+locus+indicates+unequal+contributions+of+distinct+signaling+pathways+during+development.">An allelic series at the PDGFalphaR locus indicates unequal contributions of distinct signaling pathways during development.</a> Developmental Cell. 2 (1), 103-113 (2002).</li><li>Spassky, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+steps+of+oligodendrogenesis:+insights+from+the+study+of+the+plp+lineage+in+the+brain+of+chicks+and+rodents.">The early steps of oligodendrogenesis: insights from the study of the plp lineage in the brain of chicks and rodents.</a> Developmental Neuroscience. 23 (4-5), 318-326 (2001).</li><li>Moyon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelination+Causes+Adult+CNS+Progenitors+to+Revert+to+an+Immature+State+and+Express+Immune+Cues+That+Support+Their+Migration.">Demyelination Causes Adult CNS Progenitors to Revert to an Immature State and Express Immune Cues That Support Their Migration.</a> Journal of Neuroscience. 35 (1), 4-20 (2015).</li><li>Gardner, A., Jukkola, P., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+of+rodent+hippocampal+neurons+in+culture.">Myelination of rodent hippocampal neurons in culture.</a> Nature Protocols. 7 (10), 1774-1782 (2012).</li><li>Thetiot, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+mechanism+of+early+nodal+clustering+and+myelination+onset+in+GABAergic+neurons+of+the+central+nervous+system.">An alternative mechanism of early nodal clustering and myelination onset in GABAergic neurons of the central nervous system.</a> bioRxiv. , 763573 (2019).</li><li>Dubessy, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+a+Contactin+multi-molecular+complex+secreted+by+oligodendrocytes+in+nodal+protein+clustering+in+the+CNS.">Role of a Contactin multi-molecular complex secreted by oligodendrocytes in nodal protein clustering in the CNS.</a> Glia. 67 (12), 2248-2263 (2019).</li><li>Barateiro, A., Fernandes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+oligodendrocyte+lineage+progression:+in+vitro+models+of+proliferation,+differentiation+and+myelination.">Temporal oligodendrocyte lineage progression: in vitro models of proliferation, differentiation and myelination.</a> Biochimica Et Biophysica Acta. 1843 (9), 1917-1929 (2014).</li><li>Thetiot, M., Ronzano, R., Aigrot, M. S., Lubetzki, C., Desmazi&#232;res, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Immunostaining+of+Myelinating+Organotypic+Cerebellar+Slice+Cultures.">Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures.</a> Journal of Visualized Experiments: JoVE. (145), (2019).</li><li>Mannioui, A., Zalc, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+Demyelination+and+Remyelination+in+a+Transgenic+Xenopus+laevis.">Conditional Demyelination and Remyelination in a Transgenic Xenopus laevis.</a> Methods in Molecular Biology (Clifton, N.J.). 1936, 239-248 (2019).</li></ol>

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 ...

We describe an efficient method of oligodendroglial lineage cells purification and culture. This allows to address the molecular mechanisms controlling oligodendrocytes differentiation and their interactions with neurons during the myelination process. This shaking technique is not expensive and is optimal to obtain high quantity of oligodendrocytes.Such cultures allow the production of oligodendrocyte-conditioned medium and co-cultures of oligodendrocytes with neurons. Multiple sclerosis is a disease caused by focal de-myelination in the central nervous system, secondary to oligodendrocytes loss. Oligodendrocytes culture provide a tool for better understanding how to promote re-myelination.Only oligodendroglial cell culture could provide insights into intrinsic mechanisms regulating developments and biology of oligodendrocytes. Co-cultures with neurons allow to gain insight into their impacts on neuronal physiology. To begin, use curved forceps to maintain the head of the animal at eye level.Use small surgical scissors to make a small incision at the base of the skull and cut the skull following the brain midline. Use forceps to gently peel off the two parts of the skull from the midline. Use a small surgical spoon to remove the brain from the head cavity.Put the brain in a 60-millimeter Petri dish containing ice cold PBS glucose on ice. Viewing under a stereo microscope, use fine forceps to remove the cerebellum, the brain stem, and the olfactory bulbs from the cerebral hemispheres. Use fine forceps to separate the two cerebral hemispheres.Use fine forceps to peel off the meninges. Put the cerebral cortices in a 60-millimeter Petri dish on ice. In a laminar flow hood, use a sharp scalpel to finely chop the cerebral cortices.Transfer the minced tissue into a 50-milliliter tube containing enzyme digestion medium. Incubate for 30 minutes in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide. After that, use a pipette to gently remove the enzyme digestion medium while making sure that the cortical tissue remains at the bottom of the tube.Use a P1000 micropipette to add one milliliter of DMEM 10%fetal calf serum and gently triturate the tissue. Use a 70-micron filter and the piston of a one-milliliter syringe to filter the cortical tissue into a 50-milliliter tube. Rinse the residual tissue on the inner tube wall of the 50-milliliter tube several times with DMEM 10%fetal calf serum.Fill the 50-milliliter tube with DMEM 10%fetal calf serum. Centrifuge at 423 times g for five minutes at room temperature. Carefully remove the supernatant and resuspend the cell pellet with two milliliters of DMEM 10%fetal calf serum.Gently triturate the cell pellet with the P1000 micropipette and then, with the P200 micropipette. Dilute the cell suspension with the appropriate volume of DMEM 10%fetal calf serum. Plate five milliliters of the cell suspension on a T-150 flask at a density of one times 10 to the fifth cells per square centimeter.Add 20 milliliters of warm DMEM 10%fetal calf serum to each T-150 flask. Incubate in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide. After shaking the sample overnight at 250 rpm and 37 degrees Celsius, harvest the supernatant in the flask containing mainly oligodendrocyte lineage cells, but also, some microglial cells, and plate it on non-coated 100-millimeter Petri dishes.Incubate the Petri dishes for 15 minutes in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. This allows removal of microglial cells through differential fast adhesion on the dish surface. In the meantime, fill each T-150 flask with 25 milliliters of warm, freshly prepared culture medium and incubate in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide until the second shaking.Next, transfer the supernatant from the Petri dishes into new non-coated 100-millimeter Petri dishes to allow adhesion of residual microglial cells. Incubate the Petri dishes for 15 minutes in a humidified incubator at 37 Celsius under 5%carbon dioxide. Remove the supernatant from every two Petri dishes, which contain non-adherent oligodendrocyte lineage cells, and transfer it into a 50-milliliter tube.Discard the Petri dishes plated with microglia. Centrifuge the tubes for five minutes at 423 times g. Carefully remove the supernatant and resuspend the cell pellet with one milliliter of Bottenstein-Sato medium.Pool all the pellets in a common 50-milliliter tube and adjust the volume to 20 or 30 milliliters depending on cell density with Bottenstein-Sato medium. Plate two or three precoated 100-millimeter Petri dishes with 10 milliliters of the cell suspension. Incubate in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide.Two hours later, clear the debris from the Petri dishes by refreshing all of the Bottenstein-Sato medium. Incubate for two days in the medium in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide. After two days, examine the culture under a microscope.In a laminar flow hood, under sterile conditions, renew the culture medium with 10 milliliters of warm MPB-27 low medium. Incubate for two days in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide. To harvest the OCM, collect the supernatant containing oligodendrocytes secreted factors.Filter sterilize the OCM using a 0.22-micron filter. In this protocol, oligodendrocyte lineage cells were purified from glial cultures by shaking off astrocytes and microglia. Analysis of the expression of different markers indicated that oligodendrocyte cultures were mostly pre-oligodendrocytes with 90 plus or minus 4%of O4 positive cells, 85 plus or minus 7%NG2 positive cells, and 4.7 plus or minus 2.1%of PLP positive cells, while 7.2 plus or minus 2.5%of cells were GFAP positive astrocytes.OCM produced from such cultures were added at three days in vitro to purified hippocampal neuron cultures. This treatment promotes the clustering of notal proteins. Myelination of hippocampal neurons were studied through addition of oligodendroctyes at 14 days in vitro.From 20 to 24 days in vitro, immuno staining of myelin markers, such as myelin basic protein, allowed visualization of the myelin segments and nodes of Ranvier. Core PBS is important for meninges removal. The brisk clearing is critical for oligodendroctyes viability and realizing the strengths of the flow applied with the pipette.Oligodendrocyte-conditioned medium can be added to neuronal cultures to gain insight into the impact of oligodendroctyes secreted factors on neuronal physiology. Co-cultures of oligodendroctyes with neurons can also be performed to study the myelination process.

generation-oligodendrocytes-oligodendrocyte-conditioned-medium-for-co

Research

JoVE Journal

Neuroscience

10.1K vistas.  Institut du Cerveau et de la Moelle épinière, ICM. Describimos un método eficiente de purificación y cultivo de células de linaje oligodendroglial. Esto permite abordar los mecanismos moleculares que controlan la diferenciación de oligodendrocitos y sus interacciones con las neuronas durante el proceso de mielinización. Esta técnica de agitación no es costosa y es óptima para obtener una gran cantidad de oligodendrocitos.Tales cultivos permiten la producción de medios condicionados con oligodendrocitos y co-cultivos de oligode...