<p class="jove_content">This project was funded by a grant from the Multiple Sclerosis Society of Canada to R.K. R.W.O is a recipient of a Studentship from the Multiple Sclerosis Society of Canada. S.D.R is a recipient of Post-Doctoral Fellowships from the Multiple Sclerosis Society of Canada and Canadian Institutes of Health Research.</p>


<ol>
	<li>Avellana-Adalid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+rat+oligodendrocyte+progenitors+into+proliferative+"oligospheres"+that+retain+differentiation+potential.">Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential.</a> <em style='font-style: italic'>J Neurosci Res</em>. <b>45</b>, 558-570 (1996).</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> <em style='font-style: italic'>J Cell Biol</em>. <b>85</b>, 890-902 (1980).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+control+of+cell+survival+in+the+oligodendrocyte+lineage.">Cell death and control of cell survival in the oligodendrocyte lineage.</a> <em style='font-style: italic'>Cell</em>. <b>70</b>, 31-46 (1992).</li><li>Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> <em style='font-style: italic'>Neuron</em>. <b>43</b>, 183-1891 (2004).</li><li>Camara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+axoglial+interactions+initiate+myelination+in+the+central+nervous+system.">Integrin-mediated axoglial interactions initiate myelination in the central nervous system.</a> <em style='font-style: italic'>J Cell Biol</em>. <b>185</b>, 699-712 (2009).</li><li>Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+promote+myelination+in+response+to+electrical+impulses.">Astrocytes promote myelination in response to electrical impulses.</a> <em style='font-style: italic'>Neuron</em>. <b>49</b>, 823-832 (2006).</li><li>Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> <em style='font-style: italic'>Nat Protoc</em>. <b>2</b>, 1044-1051 (2007).</li><li>Pedraza, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+characterization,+and+efficient+transfection+of+highly+pure+oligodendrocyte+precursor+cultures+from+mouse+embryonic+neural+progenitors.">Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors.</a> <em style='font-style: italic'>Glia</em>. <b>56</b>, 1339-1352 (2008).</li><li>Lewin, G. R., Ritter, A. M., Mendell, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+nerve+growth+factor+in+the+development+of+myelinated+nociceptors.">On the role of nerve growth factor in the development of myelinated nociceptors.</a> <em style='font-style: italic'>J Neurosci</em>. <b>12</b>, 1896-1905 (1992).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vitro+studies+on+the+nerve+growth+factor+(NGF)+requirement+of+neurons.+II.+Sensory+neurons.">Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons.</a> <em style='font-style: italic'>Dev Biol</em>. <b>58</b>, 106-113 (1977).</li></ol>

<p class="jove_content">No conflicts of interest declared.</p>

<p class="jove_content">This report describes a method for isolating murine OPCs for differentiation in OL-enriched cultures or OL/DRGN co-cultures. When cultured alone, the OPCs differentiate into MBP<sup>+ve</sup> OLs, producing myelin-like membranous sheets. When added to DRGN neurite beds, OLs enwrap the DRGN neurites with MBP<sup>+ve</sup> membrane. This model benefits the investigation of the complex underpinnings governing OL-mediated axonal ensheathment.</p>
<p class="jove_content">While of great value, the establishment of such cultures is technically challenging. In particular, demanding aspects include efficient tissue digestion/dissociation, maintenance of balanced culture media pH, and DRGN media changes. It is important to consider that the length of digestion, amount of tissue being digested and the amount of trituration affects the efficacy and end result of the tissue dissociation. It is not unusual for experienced researchers to obtain low cellular yields from dissociated nervous system tissues. In addition, murine OPCs tend to be sensitive to changes in the pH of the culture media, particularly under alkaline conditions. The maintenance of cultures at 8.5% CO<sub>2</sub> aims to prevent this, since OPCs appear to better tolerate slightly acidic conditions over basic. With regards to feeding DRGNs, media changes must be performed quickly as to not desiccate the neurons, however, must be gentle as to not disrupt the developing neurite bed. Abrupt media changes may dislodge the neurite bed from the substrate, and likely result in its complete dissociation from the coverslip.</p>
<p class="jove_content">The potential merit of this model system greatly overshadows its technically demanding nature. One advantage of this system is the use of post-natal mice for cell culture derivation, circumventing the need to sacrifice breeding females to harvest embryonic tissue. Another advantage is the lack of requirement for growth factors (GFs) for the expansion of OPCs. Mixed glial cultures provide an environment that supports the propagation of OPCs, presumably due to the presence of astrocyte-derived trophic factors. Other methods, such as derivation via neurospheres<sup>7,8</sup>, rely on the mitogenic properties of GFs such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) for OPC expansion. Similarly, using postnatal (P5-10) mice for DRGNs avoids the requirement of supplementing the culture media with nerve growth factor (NGF), a neurotrophic factor required for the <em>in vitro</em> survival of embryonic DRGNs <sup>9, 10</sup>. It is of interest to avoid using NGF as it negatively influences the myelinating capacity of OLs when cultured with DRGNs <sup>4</sup>. Avoiding the use of GF-supplemented media also has economic benefits, as these reagents become costly when used on large scale.</p>
<p class="jove_content">Perhaps the most important benefit of this culture model is its derivation from mouse-only tissues, thus providing the opportunities to derive both OPCs and DRGNs from the wide variety of transgenic mouse lines. This allows for the study of both DRGN and/or OPC-specific properties that govern myelination. This will be especially important for elucidating the receptor/ligand interactions regulating OL-mediated myelination of axons. In all, this technique is of great value with regards to neuroscience research due to its applications towards understanding the molecular cues underlying myelination.</p>

<table border="1">
	<tbody>
		<tr>
			<td><strong>Product Name</strong></td>
			<td><strong>Company</strong></td>
			<td><strong>Product Number</strong></td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
			<td>Multicell</td>
			<td>319-005-CL</td>
		</tr>
		<tr>
			<td>Hank's Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
		</tr>
		<tr>
			<td>Minimum Essential Media (MEM)</td>
			<td>GIBCO</td>
			<td>12360-038</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>GIBCO</td>
			<td>10091-148</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (Pen/Strep)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Poly-l-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
		</tr>
		<tr>
			<td>Human merosin purified protein (LN2)</td>
			<td>Millipore</td>
			<td>CC085</td>
		</tr>
		<tr>
			<td>Recombinant rat ciliary neurotrophic factor (CNTF)</td>
			<td>PeproTech</td>
			<td>450-50</td>
		</tr>
		<tr>
			<td>L-thyroxine</td>
			<td>Biochemika</td>
			<td>89430</td>
		</tr>
		<tr>
			<td>Holo-transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T0665</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
		</tr>
		<tr>
			<td>Bovine insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
		</tr>
		<tr>
			<td>3,3',5-Triiodo-L-thyronine</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P7505</td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
		</tr>
		<tr>
			<td>5-Fluoro-2'-deoxyuridine (FuDR)</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
		</tr>
		<tr>
			<td>Papain solution</td>
			<td>Worthington</td>
			<td>LS003126</td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>ROCHE</td>
			<td>1010159001</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352</td>
		</tr>
		<tr>
			<td>24-well tissue culture dishes</td>
			<td>Cellstar</td>
			<td>662-160</td>
		</tr>
		<tr>
			<td>T25-tissue culture flasks with vent cap</td>
			<td>Corning</td>
			<td>430639</td>
		</tr>
		<tr>
			<td>10 cm tissue culture dishes</td>
			<td>Corning</td>
			<td>430167</td>
		</tr>
		<tr>
			<td>10 cm petri dish</td>
			<td>Fisher Scientific</td>
			<td>0875713</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>ROCHE</td>
			<td>103578</td>
		</tr>
		<tr>
			<td>CellTrics 50 &mu;m filter (optional)</td>
			<td>PARTEC</td>
			<td>04-004-2327</td>
		</tr>
		<tr>
			<td>Myelin Basic Protein (MBP) Antibody</td>
			<td>AbD Serotec</td>
			<td>MCA409S</td>
		</tr>
		<tr>
			<td>NG2 antibody</td>
			<td>Millipore</td>
			<td>AB5320</td>
		</tr>
		<tr>
			<td>Myelin-Associated Glycoprotein (MAG) antibody</td>
			<td>Millipore</td>
			<td>MAB1567</td>
		</tr>
		<tr>
			<td>2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) antibody</td>
			<td>Covance</td>
			<td>SMI-91R-100</td>
		</tr>
		<tr>
			<td>Actin pan Ab-5 antibody </td>
			<td>Fitzgerald</td>
			<td>10R-A106AX</td>
		</tr>
		<tr>
			<td>Neurofilament-200 (NF) antibody</td>
			<td>Sigma-Aldrich</td>
			<td>N4142</td>
		</tr>
		<tr>
			<td>Tubulin beta-3 chain (Tuj1) antibody</td>
			<td>Millipore</td>
			<td>MAB5544</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21422</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 goat anti-rat (IgG) (H+L) secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21247</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L)-HRP conjugated  secondary antibody</td>
			<td>BioRad</td>
			<td>170-6516</td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG (H+L)-HRP conjugated secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2065</td>
		</tr>
		<tr>
			<td>4',6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
		</tr>
	</tbody>
</table>
<p class="jove_step">Media Recipes</p>
<p class="jove_content"><strong>100X OL-Supplement* </strong></p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Ingredient</strong></td>
			<td><strong>Amount to add</strong></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>1.02 g</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>0.6 mg</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>161 mg</td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>0.05 mg</td>
		</tr>
		<tr>
			<td>3,3',5-Triiodo-L-thyronine</td>
			<td>4 mg</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">*Store at -80&deg;C in 250 &mu;L aliquots</p>
<p class="jove_content"><strong>OL media</strong></p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Ingredient</strong></td>
			<td><strong>Amount to add</strong></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>23.75 mL</td>
		</tr>
		<tr>
			<td>100X OL-Supplement</td>
			<td>250 &mu;L</td>
		</tr>
		<tr>
			<td>Bovine insulin (from 1 mg/mL stock)</td>
			<td>125 &mu;L</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>250 &mu;L</td>
		</tr>
		<tr>
			<td>Holo-transferrin (from 33 mg/mL stock)</td>
			<td>37.5 &mu;L</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>500 &mu;L</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>125 &mu;L</td>
		</tr>
		<tr>
			<td>CNTF (from 50 ng/&mu;L stock)</td>
			<td>25 &mu;L</td>
		</tr>
	</tbody>
</table>
<p class="jove_content"><strong>Mixed glial culture media (made up in DMEM)</strong></p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Ingredient</strong></td>
			<td><strong>Final concentration </strong></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Pen/Strep (0.33% from stock)</td>
			<td>33 units/mL Penicillin and 33 &mu;g/mL Streptomycin</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>1%</td>
		</tr>
	</tbody>
</table>
<p class="jove_content"><strong>DRGN media (made up in DMEM)</strong></p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Ingredient</strong></td>
			<td><strong>Final concentration </strong></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Pen/Strep (1% from stock)</td>
			<td>100 units/mL Penicillin and 100 &mu;g/mL Streptomycin</td>
		</tr>
	</tbody>
</table>
<p class="jove_step">Digestion Solution Recipes:</p>
<p class="jove_content"><strong>OPC papain solution (made up in MEM)</strong></p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Ingredient</strong></td>
			<td><strong>Final concentration </strong></td>
		</tr>
		<tr>
			<td>Papain solution</td>
			<td>1.54 mg/mL</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>360 &mu;g/mL</td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>60 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
<p class="jove_content"><strong>DRG papain solution (made up in HBSS)</strong></p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Ingredient</strong></td>
			<td><strong>Final concentration </strong></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>1.54 mg/mL</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>360 &mu;g/mL</td>
		</tr>
	</tbody>
</table>
<p class="jove_content"><strong>DRG Collagenase A solution (made up in HBSS)</strong></p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Ingredient</strong></td>
			<td><strong>Final concentration </strong></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>4 mg/mL</td>
		</tr>
	</tbody>
</table>

derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues

<p class="jove_content">Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present <em>in vivo</em>. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. </p>
<p class="jove_content">Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification <sup>1-3</sup>. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis <sup>2</sup> is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. </p>
<p class="jove_content">While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published <sup>4-6</sup>, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.</p>


Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

<p class="jove_content">This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).</p>

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

35.9K visualizações.  Ottawa Hospital Research Institute. Este artigo descreve métodos para derivar populações enriquecidas de células precursoras de oligodendrócitos murino (OPCs) em cultura primária, que se diferenciam para produzir oligodendrócitos maduros (MQO). Além disso, este relatório descreve as técnicas para produzir murino myelinating co-culturas semeando OPCs do mouse sobre uma cama de neurite do mouse neurônios do gânglio da raiz dorsal (DRGNs).