Name: derivation of leptomeninges explant cultures from postmortem human brain donors
Uploaded: 2017-01-21T13:00:00.000+00:00
Duration: 5 min 18 s
Description: Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Development of this protocol was funded by private donations directed to the Parkinson's Institute Brain Donation Program.

The brain donation registration includes documentation by the registrant of their intent to donate. The autopsy permission for tissue retrieval is provided by the next of kin as permitted by law. Research studies using collected autopsy specimens are reviewed by the institutional review board (IRB) to ensure compliance with Health Insurance Portability and Accountability Act (HIPAA) regulations.
NOTE: Leptomeninges samples are collected by the brain dissector or neuropathologist during a brain dissection and are stored in 50 mL conical tubes containing 25-30 mL meninges growth media. The sample is stored at 4 &#176;C until sample preparation. The processing should be performed as soon as possible as the viability of cells decreases with time post mortem.
1. Set-up before Starting the Leptomeninges Dissection
NOTE: Steps 1.1 - 1.3 are to be performed inside a biosafety cabinet.
<ol>
	<li>Prepare meninges growth media by combining 375 mL of Dulbecco&#39;s Modified Eagle Media, 100 mL of fetal bovine serum, 5 mL 10 mM non-essential amino acids, 5 mL 200 mM L-alanyl-L-glutamine, 5 mL 100 mM sodium pyruvate, 5 mL 10,000 U/mL penicillin/streptomycin and 5 mL 250 &#181;g/mL Amphotericin B in 500 mL filter unit. Filter and mix. Use media for up to four weeks.</li>
	<li>Place all materials in the biosafety cabinet before starting the dissection: 4x Petri dishes, 2x 6-well plates, 2x scalpels, 15-20 sterilized 15-mm cover slips, phosphate buffer saline (PBS), serological pipettes, aspirating pipettes.</li>
	<li>Add 1 mL of sterile-autoclaved 0.1% gelatin solution to each well of a 6-well plate. Set the plate aside for 30-60 min. Prepare 2x 6-well plates.</li>
</ol>
2. Preparation of Leptomeninges Sample
NOTE: Steps 2.1 - 2.3 are to be performed inside a biosafety cabinet.
<ol>
	<li>Add approximately 10 mL of PBS to a sterile 10-cm tissue culture dish and place the meninges sample in the dish. Using forceps, gently wash leptomeninges with PBS to remove blood.</li>
	<li>Transfer the meninges into a new 10-cm tissue culture dish containing 10 mL of fresh PBS and continue washing. Repeat as necessary to remove as much blood as possible.</li>
	<li>Using two scalpels, cut leptomeninges tissue into an approximately 6 cm x 6 cm large piece.</li>
	<li>Place lid on a culture dish and transfer to a dissecting microscope in a horizontal laminar flow hood.</li>
</ol>
3. Dissection of Leptomeninges Tissue
NOTE: Steps 3.1 - 3.3 are to be performed inside a horizontal laminar flow hood using a dissecting microscope.
<ol>
	<li>Remove blood vessels from a region on the leptomeninges piece (step 2.3) large enough to create at least 20-25 3 mm x 3 mm pieces. Hold the leptomeninges in place with one scalpel and with the other, use a gentle and shallow scraping motion to separate the vessels.</li>
	<li>Cut small squares of tissue from the prepared region. Cut in a stepwise process by first making larger pieces and then slicing those into smaller sections.</li>
	<li>Use one scalpel to hold the tissue in place and another scalpel to cut the tissue with a rocking motion. Due to the consistency of the tissue, it is difficult to slide the scalpels against each other. This will only tear the tissue apart and results in ragged edges.</li>
	<li>Continue cutting the pieces in half until there are about 20-25 3 mm x 3 mm pieces.</li>
</ol>
4. Transfer of Dissected Meninges Pieces onto Tissue Culture Plates
NOTE: Step 4 is to be performed inside a biosafety cabinet.
<ol>
	<li>Aspirate gelatin from 6-well plate (from step 1.3). Add 1 mL of meninges growth media to each well. Using sterile forceps, place 3-4 biopsy pieces into each well.</li>
</ol>
5. Placement of Cover Slips over Meninges Pieces
NOTE: Steps 5.1 - 5.2 are to be performed inside a horizontal laminar flow hood using a dissecting microscope.
<ol>
	<li>Cover the pieces with 1-2 sterile 15-mm circular coverslips per well.</li>
	<li>Press down firmly with forceps to ensure the pieces are touching the bottom of the plate. This allows attachment of tissue pieces to the bottom of the well.</li>
	<li>Place the 6-well plate into a 37 &#176;C, 5% CO2 incubator.</li>
</ol>
6. Cell Culture Maintenance
NOTE: Steps 6.1 - 6.3 are to be performed inside a biosafety cabinet. Handle culture dishes with care as coverslips should not move and dislodge the meninges pieces.
<ol>
	<li>After 2 days in culture, add an additional 1 mL of fresh meninges growth media to each well.</li>
	<li>At Day 7, carefully aspirate the media and add 2 mL of fresh media to each well.</li>
	<li>After Day 9, continue to change growth media every other day until culture becomes confluent.</li>
</ol>
7. Passaging
NOTE: All steps are to be performed inside a biosafety cabinet. Once meningeal fibroblasts have migrated out of the meningeal tissue pieces and start to grow towards the edges of the culture vessel, expand meningeal fibroblasts into larger culture dish.
<ol>
	<li>Before starting the passaging, coat the culture dish with 1% gelatin for 15-30 min.</li>
	<li>Aspirate media from 6-well dish and wash cells with 2 mL PBS.</li>
	<li>Add 1 mL of trypsin/EDTA to each well and incubate at 37 &#176;C for 5-10 min. It is not necessary to remove the cover slips or the meningeal tissue for this step.</li>
	<li>When cells are rounded and start to detach, add 2 mL of culture media.</li>
	<li>Pipette cells up and down to completely disrupt all clumps and transfer the cell solution to larger culture dish.</li>
	<li>Use a ratio of 1:3 to 1:4 to combine cells from three 6-well dishes into one T75 culture dish.</li>
	<li>Once meningeal fibroblasts cells are confluent in T75 culture flasks, trypsinize the cells, and cryopreserve in cryopreservation media at 1x106 cells/mL per vial. One T75 culture flask with confluent meningeal fibroblasts contains about 5-7 million cells.</li>
</ol>
8. Characterization of the Meningeal Fibroblasts by Immunostaining
NOTE: Before starting the procedure, prepare 4% paraformaldehyde solution, 5% normal goat serum in PBS (blocking buffer), 0.3% Triton X-100 in PBS (permeabilization buffer), primary and secondary antibody solutions, and 1 &#181;g/mL of Hoechst 33342 diluted in PBS from 10 mg/mL stock.
<ol>
	<li>Seed 15,000 to 30,000 meningeal fibroblasts in 8-well chamber slide coated with 0.1% gelatin.</li>
	<li>After 24 h when the cells are attached, fix cells in 100 &#181;L 4% paraformaldehyde per well for 10 min at room temperature. Wash the wells 3 times with 1x PBS.</li>
	<li>Permeabilize the cells with 150 &#181;L of 0.3% Triton X-100 for 5 min at room temperature. Wash the wells 3 times with 1x PBS.</li>
	<li>Add 200 &#181;L blocking solution (PBS + 5% normal goat serum) for 1 h at room temperature.</li>
	<li>Aspirate the blocking solution, and add 150 &#181;L of desired primary antibodies diluted in blocking solution. For double-staining, prepare working dilutions of primary antibodies in separate centrifuge tubes on ice as follows:
	<ol>
		<li>Prepare 1:300 dilution of anti-Fibronectin + 1:200 dilution of anti-nestin in blocking solution. Prepare 1:250 dilution of anti-SERPINH1 + 1:1,000 dilution of anti-TUJ1 in blocking solution. Prepare 1:250 dilution of anti-SERPINH1 + 1:100 dilution of anti-SOX2 in blocking solution</li>
	</ol>
	</li>
	<li>Incubate at 4 &#176;C overnight.</li>
	<li>Wash the wells 3 times with 1x PBS.</li>
	<li>Prepare a working 1:400 dilution of fluorescently labeled secondary antibodies goat anti-rabbit (green; Ex/Em2 495/519 nm) and goat anti-mouse (red; Ex/Em2 590/617 nm) in blocking solution and add 100 &#181;L to each well. Incubate for 1 h, in the dark, at room temperature. Then wash wells once with 1x PBS.</li>
	<li>Add 100 &#181;L of 1 &#181;g/mL Hoechst 33342 (blue; Ex/Em2 358/461 nm) solution to each well and incubate in the dark for 3 min. Wash wells 3 times with 1x PBS, leaving in the final 1x PBS wash in the well, and cover the slide with aluminum foil.</li>
	<li>Analyze cells under fluorescent microscope.</li>
</ol>

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E., 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=Induction+of+pluripotent+stem+cells+from+autopsy+donor-derived+somatic+cells.">Induction of pluripotent stem cells from autopsy donor-derived somatic cells.</a> Neurosci Lett. 502 (3), 219-224 (2011).</li><li>Hjelm, B. E., 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=In+vitro-differentiated+neural+cell+cultures+progress+towards+donor-identical+brain+tissue.">In vitro-differentiated neural cell cultures progress towards donor-identical brain tissue.</a> Hum Mol Genet. 22 (17), 3534-3546 (2013).</li><li>Meske, V., Albert, F., Wehser, R., Ohm, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+autopsy-derived+fibroblasts+as+a+tool+to+study+systemic+alterations+in+human+neurodegenerative+disorders+such+as+Alzheimer's+disease--methodological+investigations.">Culture of autopsy-derived fibroblasts as a tool to study systemic alterations in human neurodegenerative disorders such as Alzheimer's disease--methodological investigations.</a> J Neural Transm (Vienna). 106 (5-6), 537-548 (1999).</li><li>Bliss, L. 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=Use+of+postmortem+human+dura+mater+and+scalp+for+deriving+human+fibroblast+cultures.">Use of postmortem human dura mater and scalp for deriving human fibroblast cultures.</a> PLoS One. 7 (9), e45282 (2012).</li><li>Sproul, A. 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=Generation+of+iPSC+lines+from+archived+non-cryoprotected+biobanked+dura+mater.">Generation of iPSC lines from archived non-cryoprotected biobanked dura mater.</a> Acta Neuropathol Commun. 2, 4 (2014).</li><li>Adler, C. H., 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=Low+clinical+diagnostic+accuracy+of+early+vs+advanced+Parkinson+disease:+clinicopathologic+study.">Low clinical diagnostic accuracy of early vs advanced Parkinson disease: clinicopathologic study.</a> Neurology. 83 (5), 406-412 (2014).</li><li>Hughes, A. J., Daniel, S. E., Kilford, L., Lees, A. 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J., Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisystem+Lewy+body+disease+and+the+other+parkinsonian+disorders.">Multisystem Lewy body disease and the other parkinsonian disorders.</a> Nature Genetics. 47 (12), 1378-1384 (2015).</li><li>Vangipuram, M., Ting, D., Kim, S., Diaz, R., Sch&#252;le, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+punch+biopsy+explant+culture+for+derivation+of+primary+human+fibroblasts.">Skin punch biopsy explant culture for derivation of primary human fibroblasts.</a> J Vis Exp. (77), e3779 (2013).</li><li>Byers, B., 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=SNCA+triplication+Parkinson's+patient's+iPSC-derived+DA+neurons+accumulate+alpha-synuclein+and+are+susceptible+to+oxidative+stress.">SNCA triplication Parkinson's patient's iPSC-derived DA neurons accumulate alpha-synuclein and are susceptible to oxidative stress.</a> PLoS One. 6 (11), e26159 (2011).</li><li>Sanders, L. H., 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=LRRK2+mutations+cause+mitochondrial+DNA+damage+in+iPSC-derived+neural+cells+from+Parkinson's+disease+patients:+reversal+by+gene+correction.">LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.</a> Neurobiol Dis. 62, 381-386 (2014).</li><li>Beevers, J. 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The authors have nothing to disclose.

This protocol describes a simple and robust protocol to derive a meningeal fibroblast culture from human postmortem leptomeninges collected in conjunction with a brain donation. There are very few descriptions of protocols to derive cell cultures from postmortem human material. Two studies describe skin-derived fibroblast cultures7,8,9, one study describes dura samples10, and another describes non-cryopre...

Meninges consist of three membranes that protect the brain: dura mater, arachnoid and pia mater. More recently, it has been recognized that meninges also play an important role in brain development and brain homeostasis1. Meninges are derived from mesenchymal and neural crest-derived cells and interestingly, it has been shown that progenitor cells residing in meninges can give rise to neurons in vitro and after transplantation in vivo2,3,4. Meninges cultures have been also successfully used as feeder layers, as they possess stromal cell-derived inducing activity for differentiation of embryonic stem cells into dopaminergic neurons5. Moreover, leptomeninges have the potential to directly differentiate into neurons, astrocytes, and oligodendrocytes under ischemic conditions6.

For this protocol, human postmortem meninges samples are collected from the arachnoid and pia mater, collectively called the leptomeninges, and are procured as part of a human brain donation for research purposes. Dissection of the brain is performed within 24 h of death and the leptomeninges sample is placed in cold growth media for further processing within the next 6-8 h as illustrated here in this protocol.
This protocol describes the dissection and preparation of human meninges samples for the development of patient primary leptomeninges cell culture. The tissue is cut into 25-30 pieces of approximately 3 mm x 3 mm squares. Three pieces are placed in each 6-well gelatin-coated well and held down with round glass coverslips. The meninges dissection takes about 25-35 min. The main challenge with this culture is sterility as the brain procurement, transport, and dissection are generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B and use multi-well dishes to separately culture tissue pieces.
Outgrowth of meningeal fibroblasts occurs usually within the first week. Media is changed every two to three days until cells are confluent and the cells are enzymatically passaged. The meningeal fibroblasts are cryopreserved at 1 million cells per mL/vial in cryopreservation media. With this protocol, 20-30 million meningeal fibroblasts can be derived in 6-8 weeks for cryopreservation. Downstream applications of these meningeal fibroblasts are primary cultures for disease research, direct neuronal differentiation or derivation of induced pluripotent stem cells from the leptomeninges for understanding of disease mechanisms and for drug development.

<table><tbody><tr><td>Corning Petri dishes</td><td>Fisher Scientific</td><td>351029</td><td></td></tr><tr><td>Nunc 6-well plate</td><td>Fisher Scientific</td><td>14-832-11</td><td></td></tr><tr><td>15-mm cover slips</td><td>Fisher Scientific</td><td>12-545-83 15CIR-1D</td><td></td></tr><tr><td>Scalpels, sterile blade, No. 15</td><td>Miltex</td><td>4-415</td><td></td></tr><tr><td>Curved precision tip forceps</td><td>Fisher Scientific</td><td>16-100-122</td><td></td></tr><tr><td>Serological pipettes</td><td>Fisher Scientific</td><td>13-678-11E</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Fisher Scientific</td><td>22-230490</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G1890-100G</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Fisher Scientific</td><td>SH30264.02</td><td></td></tr><tr><td>Corning 500 mL filter unit</td><td>Fisher Scientific</td><td>430770</td><td>Combine media components and filter.</td></tr><tr><td>Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm&lt;sup&gt;2&lt;/sup&gt;&amp;nbsp;</td><td>Thermo Scientific</td><td>178883</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Growth Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hyclone DMEM</td><td>Fisher Scientific</td><td>SH30081.02</td><td></td></tr><tr><td>Hyclone FBS</td><td>Fisher Scientific</td><td>SH30910.03</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Thermo Fisher</td><td>11140-050</td><td></td></tr><tr><td>GlutaMAX Supplement (100x)</td><td>Thermo Fisher</td><td>35050-061</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Thermo Fisher</td><td>11360-070</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Amphotericin B (Yellow Solution/250 &amp;micro;g/mL)</td><td>Fisher Scientific</td><td>BP264520</td><td></td></tr><tr><td>Bambanker Freeze 120 mL</td><td>Fisher Scientific</td><td>NC9582225</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Fibronectin Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>8 well chamber slides&amp;nbsp;</td><td>Fisher Scientific</td><td>1256518</td><td></td></tr><tr><td>20% paraformaldehyde&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>15713</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>100% Glycerol&amp;nbsp;</td><td>BioRad</td><td>9455</td><td></td></tr><tr><td>100% normal goat serum&amp;nbsp;</td><td>Fisher Scientific</td><td>101098-382</td><td></td></tr><tr><td>Anti-Fibronectin antibody [F1]</td><td>Abcam</td><td>ab32419</td><td>1:300 dilution in blocking solution</td></tr><tr><td>Anti-SERPINH1</td><td>Sigma</td><td>S5950-200ul</td><td>1:250 dilution in blocking solution</td></tr><tr><td>Anti-SOX2</td><td>Millipore</td><td>MAB4343</td><td>1:100 dilution in blocking solution</td></tr><tr><td>Anti-Nestin</td><td>Millipore</td><td>MAB5326</td><td>1:200 dilution in blocking solution</td></tr><tr><td>Anti-TUJ1</td><td>Covance</td><td>MMS-435P</td><td>1:1,000 dilution in blocking solution</td></tr><tr><td>Alexa Fluor 488 anti-rabbit</td><td>Thermo Fisher</td><td>A11029</td><td>1:400 dilution in blocking solution; (green channel; Ex/Em2 495/519 nm)&amp;nbsp;</td></tr><tr><td>Alexa Fluor 555 anti-mouse</td><td>Thermo Fisher</td><td>A21424</td><td>1:400 dilution in blocking solution; (red channel; Ex/Em2 590/617 nm)&amp;nbsp;</td></tr><tr><td>Hoechst 33342 stain</td><td>Thermo Fisher</td><td>H3570</td><td>dilute to a final concentration of 1.0 &amp;mu;g/mL; (blue channel; Ex/Em2 358/461 nm)</td></tr><tr><td colspan="4">Suppliers are suggestions, similar products from alternative vendors can be used as well.</td></tr></tbody></table>

derivation of leptomeninges explant cultures from postmortem human brain donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

When the leptomeninges processing protocol has been successful, outgrowth of meningeal fibroblasts is first observed three to nine days after dissection, although this can depend on the length of post-mortem interval for the brain. Figure 1 demonstrates meningeal fibroblast cultures of four different donors. Figure 1A shows a leptomeninges piece held down by a glass cover slip (dark diagonal line) and fibroblast outgrowth around the tissue four days after...

Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

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8.2K Views.  Parkinson’s Institute and Clinical Center. The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Video: Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

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

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 in vivo. 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. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. 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 2 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. 
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 4-6, 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.

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).

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

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Murine Model for Studying Leptomeningeal Disease Using Circulating Tumor Cells

Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma&#45;Associated Leptomeningeal Disease

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the meninges, the membrane layers that protect the brain and the spinal cord. Once established, the prognosis for M-LMD patients is dismal, with overall survival ranging from weeks to months. This is primarily due to a paucity in our understanding of the disease and, as a consequence, the availability of effective treatment options. Defining the underlying biology of M-LMD will significantly improve the ability to adapt available therapies for M-LMD treatment or design novel inhibitors for this universally fatal disease. A major barrier, however, lies in obtaining sufficient quantities of CTCs from the patient-derived CSF (CSF-CTCs) to conduct preclinical experiments, such as molecular characterization, functional analysis, and in vivo efficacy studies. Culturing CSF-CTCs ex vivo has also proven to be challenging. To address this, a novel protocol for the culture of patient-derived M-LMD CSF-CTCs ex vivo and in vivo is developed. The incorporation of conditioned media produced by human meningeal cells (HMCs) is found to be critical to the procedure. Cytokine array analysis reveals that factors produced by HMCs, such as insulin-like growth factor-binding proteins (IGFBPs) and vascular endothelial growth factor-A (VEGF-A), are important in supporting CSF-CTC survival ex vivo. Here, the usefulness of the isolated patient-derived CSF-CTC lines is demonstrated in determining the efficacy of inhibitors that target the insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, the ability to intrathecally inoculate these cells in vivo to establish murine models of M-LMD that can be employed for preclinical testing of approved or novel therapies is shown. These tools can help unravel the underlying biology driving CSF-CTC establishment in the meninges and identify novel therapies to reduce the morbidity and mortality associated with M-LMD.

Author Spotlight: Assessing the Potential of Circulating Tumor Cells in Leptomeningeal Disease Research

This article describes a protocol for the propagation of cerebral spinal fluid-circulating tumor cells (CSF-CTCs) collected from patients with melanoma-associated leptomeningeal disease (M-LMD) to develop preclinical models to study M-LMD.

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the ...

Cancer Research

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Immunology and Infection

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find preventive and curative measures. The most effective way to achieve this is through direct examination of diseased and healthy brain tissue. The authors present a protocol to obtain, process, characterize and store good quality brain tissue donated by individuals registered in an antemortem brain donation program. The donation program includes a face-to-face empathic approach to people, a collection of complementary clinical, biological, social and lifestyle information and serial multi-dimensional assessments over time to track individual trajectories of normal aging and cognitive decline. Since many neurological diseases are asymmetrical, our brain bank offers a unique protocol for slicing fresh specimens. Brain sections of both hemispheres are alternately frozen (at -80 &#176;C) or fixed in formalin; a fixed slice on one hemisphere corresponds to a frozen one on the other hemisphere. With this approach, a complete histological characterization of all frozen material can be obtained, and omics studies can be performed on histologically well-defined tissues from both hemispheres thus offering a more complete assessment of neurodegenerative disease mechanisms. Correct and definite diagnosis of these diseases can only be achieved by combining the clinical syndrome with the neuropathological evaluation, which often adds important etiological clues necessary to interpret the pathogenesis. This method can be time consuming, expensive and limited as it only covers a limited geographical area. Regardless of its limitations, the high degree of characterization it provides can be rewarding. Our ultimate goal is to establish the first Italian Brain Bank, all the while emphasizing the importance of neuropathologically verified epidemiological studies.

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find ...

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

Embryonic Mouse Brain Mixed Cell Cultures to Study Infection and Innate Immunity

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Author Spotlight: Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

Establishing In Vitro LLEC Cultures Using Magnetic Cell Selection

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

Preparing Free-Floating Slice Cultures from an Adult Human Brain

Source: Fernandes, A., et al. <a href="https://app.jove.com/v/59845">Short-Term Free-Floating Slice Cultures from the Adult Human Brain.</a> J. Vis. Exp. (2019).
This video demonstrates a technique for preparing free-floating slice cultures from the adult human brain. Thin slices are generated from a brain section using a vibratome. These slices are then suspended in a culture medium supplemented with growth factors to maintain tissue viability.

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