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

Das Gehirn Spende Anmeldung umfasst die Dokumentation vom Registranten ihrer Absicht zu spenden. Die Autopsie Erlaubnis für Gewebeentnahme wird durch die nächsten Angehörigen zur Verfügung gestellt, wie dies gesetzlich zulässig ist. Wissenschaftliche Studien gesammelt Autopsieproben verwendet werden von der Institutional Review Board (IRB) überprüft, um die Einhaltung Health Insurance Portability and Accountability Act (HIPAA) Vorschriften. HINWEIS: Leptomeningen Proben werden durch das Gehirn Dissektor oder neuropathologist während einer Gehirnsezierung gesammelt und in 50 ml konischen Röhrchen, die 25-30 ml Meningen Wachstumsmedium gespeichert. Die Probe wird bei 4ºC bis zur Probenvorbereitung aufbewahrt. Die Verarbeitung sollte so schnell wie möglich durchgeführt werden, da die Lebensfähigkeit von Zellen mit der Zeit post mortem abnimmt. 1. Set-up, bevor die Leptomeningen Dissection starten Hinweis: Die Schritte 1,1-1,3 sind in einem Biosicherheitsschrank durchgeführt werden. <ol><li> Bereiten Meningen Wachstumsmedien durch die Kombination 375 ml Dulbeccos modifiziertem Eagle-Medium, 100 ml fötalem Rinderserum, 5 ml 10 mM nicht-essentielle Aminosäuren, 5 ml 200 mM L-Alanyl-L-Glutamin, 5 ml 100 mM Natriumpyruvat, 5 ml 10.000 U / ml Penicillin / Streptomycin und 5 ml 250 ug / ml Amphotericin B in 500 ml-Filtereinheit. Filter und mischen. Verwenden Sie Medien für bis zu vier Wochen. </li><li> Legen Sie alle Materialien, die in der Biosicherheitsschrank vor der Präparation beginnen: 4x Petrischalen, 2x 6-Well-Platten, 2x Skalpelle, 15-20 sterilisierten 15-mm-Deckgläser, Phosphatpuffer-Salzlösung (PBS), serologischen Pipetten, Absaugen Pipetten. </li><li> 1 ml steriles autoklavierte 0,1% ige Gelatinelösung zu jeder Vertiefung einer Platte mit 6 Vertiefungen. Stellen Sie den Teller zur Seite 30-60 min. Bereiten Sie 2x 6-Well-Platten. </li></ol> 2. Herstellung von Leptomeningen Proben Hinweis: Die Schritte 2,1-2,3 sind in einem Biosicherheitswerkbank durchgeführt werden. <ol><li> In etwa 10 ml PBSeine sterile 10-cm-Gewebekulturschale und die Meningen Probe in die Schüssel legen. Mit einer Pinzette vorsichtig waschen Leptomeningen mit PBS Blut zu entfernen. </li><li> Übertragen Sie die Meningen in eine neue 10-cm-Gewebekulturschale mit 10 ml frischem PBS und Waschen fortzusetzen. Bei Bedarf wiederholen, so viel Blut wie möglich zu entfernen. </li><li> Mit Hilfe von zwei Skalpelle schneiden Leptomeningen Gewebe in einem ca. 6 cm x 6 cm großes Stück. </li><li> Deckel auf einer Kulturschale und in ein Präpariermikroskop in einer horizontalen laminaren Strömungshaube. </li></ol> 3. Dissection von Leptomeningen Tissue Hinweis: Die Schritte 3,1-3,3 sind in einer horizontalen Laminar-Flow-Haube durchgeführt werden, um ein Binokular mit. <ol><li> Entfernen Blutgefäße aus einem Bereich auf der Leptomeningen Stück (Schritt 2.3) groß genug, um mindestens 20 bis 25 März mm x 3 mm Stücke zu schaffen. Halten Sie die Leptomeningen an Ort und Stelle mit einem Skalpell und mit dem anderen, verwenden Sie eine sanfte und flache Schaben motion die Gefäße zu trennen. </li><li> Schneiden Sie kleine Quadrate von Gewebe aus dem vorbereiteten Region. Schneiden Sie in einem schrittweisen Prozess durch erste größere Stücke zu machen und dann diejenigen in kleinere Abschnitte schneiden. </li><li> Verwenden Sie ein Skalpell, das Gewebe in Position zu halten und ein weiteres Skalpell das Gewebe mit einer Schaukelbewegung zu schneiden. Aufgrund der Konsistenz des Gewebes, ist es schwierig, die Skalpelle, gegeneinander zu gleiten. Dies wird nur das Gewebe zerreißen und führt in unregelmäßigen Kanten. </li><li> Weiter, um die Stücke zu halbieren, bis es ca. 20-25 3 mm x 3 mm große Stücke. </li></ol> 4. Übertragung von Dissected Meninges Stück auf Gewebekulturplatten HINWEIS: Schritt 4 ist im Inneren eines biologischen Sicherheit Schrank durchgeführt werden. <ol><li> Absaugen Gelatine aus 6-Well-Platte (aus Schritt 1.3). 1 mL Hirnhaut Wachstumsmedien in jede Kammer. Mit einer sterilen Pinzette, legen 3-4 Biopsie Stücke in jede Vertiefung. </li></ol> 5. Platzierung der Deckgläser über Meninges Pieces Hinweis: Die Schritte 5,1-5,2 sind in einer horizontalen Laminar-Flow-Haube durchgeführt werden, um ein Binokular mit. <ol><li> Decken Sie die Stücke mit 1-2 sterilen 15-mm-Kreisdeck pro Vertiefung. </li><li> Drücken Sie fest mit einer Pinzette die Stücke berühren den Boden der Platte zu gewährleisten. Dies ermöglicht Befestigung von Gewebestücken an den Boden des Bohrlochs. </li><li> Legen Sie die 6-Well - Platte in eine 37 ° C, 5% CO 2 Inkubator. </li></ol> 6. Zellkultur Wartung Hinweis: Die Schritte 6,1-6,3 sind in einem Biosicherheitswerkbank durchgeführt werden. Behandeln Sie Kulturschalen mit Sorgfalt als Deck sollten nicht die Meningen Stücke bewegen und entfernen. <ol><li> Nach 2 Tagen in Kultur zusätzlich 1 ml frisches Meningen Wachstumsmedien in jede Vertiefung hinzu. </li><li> Am 7. Tag absaugen sorgfältig die Medien und fügen Sie 2 ml frisches Medium zu jeder Vertiefung. </li><li> Nach dem Tag 9, weiterhin Wachstumsmedien wechseln alleanderen Tag bis Kultur wird konfluent. </li></ol> 7. Passagierung HINWEIS: Alle Schritte in einem Biosicherheitsschrank durchgeführt werden sollen. Sobald meningealen Fibroblasten wurden aus den meningealen Gewebestücke migriert und beginnen zu den Rändern des Kulturgefäßes zu wachsen, meningealen Fibroblasten in größere Kulturschale erweitern. <ol><li> Vor dem Start der Passagierung Beschichtung der Kulturschale mit 1% Gelatine für 15-30 min. </li><li> Absaugen Medien von 6-Well-Platte und waschen Sie die Zellen mit 2 ml PBS. </li><li> 1 ml Trypsin / EDTA zu jeder Vertiefung und Inkubation bei 37 ° C für 5-10 min. Es ist nicht erforderlich, die Deckgläser oder die meningealen Gewebe für diesen Schritt zu entfernen. </li><li> Wenn die Zellen sind gerundet und beginnen zu lösen, fügen Sie 2 ml Kulturmedien. </li><li> Pipette Zellen nach oben und unten, um vollständig alle Klumpen stören und die Zelllösung zu größeren Kulturschale übertragen. </li><li> Verwenden, um ein Verhältnis von 1: 3 bis 1: 4 zu Zellen von drei 6-Well-Schalen in kombinierenzu einer T75 Kulturschale. </li><li> Sobald Zellen meningealen Fibroblasten in T75 Kulturflaschen konfluent sind, trypsinize die Zellen, und in Kryokonservierung Medien bei 1x10 6 Zellen / ml pro Fläschchen Kryokonservierung. Ein T75 Kulturflasche mit konfluenten meningeal Fibroblasten enthält etwa 5-7 Millionen Zellen. </li></ol> 8. Charakterisierung der Meningeale Fibroblasten durch Immunostaining HINWEIS: Vor Beginn des Verfahrens, Herstellung von 4% Paraformaldehydlösung, 5% normalem Ziegenserum in PBS (Blocking-Puffer), 0,3% Triton X-100 in PBS (Permeabilisierung Puffer), primäre und sekundäre Antikörperlösungen und 1 &amp; mgr; g / ml Hoechst 33342 in PBS von 10 mg / ml Stamm verdünnt. <ol><li> Seed 15.000 bis 30.000 meningealen Fibroblasten in 8-Well-Kammerobjektträger mit 0,1% Gelatine beschichtet. </li><li> Nach 24 Stunden, wenn die Zellen gebunden sind, fixieren Zellen in 100 &amp; mgr; l 4% Paraformaldehyd pro Vertiefung für 10 Minuten bei Raumtemperatur. Waschen Sie die Vertiefungen 3 mal mit 1x PBS. </li><li> Permeabilisierung der Zellen mit 150 &amp; mgr; l 0,3% Triton X-100 für 5 min bei Raumtemperatur. Waschen Sie die Vertiefungen 3 mal mit 1x PBS. </li><li> Mit 200 &amp; mgr; l Blockierungslösung (PBS + 5% normales Ziegenserum) für 1 h bei Raumtemperatur. </li><li> Saugen Sie das Blockierungslösung, und fügen Sie 150 ul der gewünschten Primär in Blockierungslösung verdünnt Antikörper. Für Doppel-Färbung, bereiten Verdünnungen von primären Antikörpern in separaten Zentrifugenröhrchen auf Eis arbeitet wie folgt: <ol><li> Vorbereitung 1: 300 Verdünnung von anti-Fibronectin + 1: 200-Verdünnung von Anti-Nestin in Blockierlösung. Bereiten Sie 1: 250 Verdünnung von Anti-SERPINH1 + 1: 1000 Verdünnung von Anti-TuJ1 in Blocking-Lösung. Vorbereitung 1: 250 Verdünnung von anti-SERPINH1 + 1: 100-Verdünnung von anti-SOX2 in Blockierungslösung </li></ol></li><li> über Nacht bei 4 ° C inkubieren. </li><li> Waschen Sie die Vertiefungen 3 mal mit 1x PBS. </li><li> Bereiten Sie eine Arbeits 1: 400 Verdünnung von fluoreszenzmarkierten Sekundärantikörper Ziege anti-Kaninchen (grün; Ex / Em 2 </sup&gt; 495/519 nm) und Ziege anti-Maus (rot; Ex / Em 2 590/617 nm) in Blocking - Lösung und mit 100 &amp; mgr; l zu jeder Vertiefung. Inkubieren für 1 h im Dunkeln bei Raumtemperatur. Dann waschen Brunnen einmal mit 1x PBS. </li><li> Je 100 &amp; mgr; l von 1 ug / ml Hoechst 33342 (blau; Ex / Em 2 358/461 nm) Lösung in jede Vertiefung und Inkubation für 3 min im Dunkeln. Waschen Sie Wells 3 mal mit 1x PBS, in der auch in der letzten 1x PBS waschen lassen, und decken Sie die Folie mit einer Aluminiumfolie. </li><li> Analysieren Sie die Zellen unter dem Fluoreszenzmikroskop. </li></ol>

<ol>
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The authors have nothing to disclose.

Dieses Protokoll beschreibt einen einfachen und robusten Protokoll eine meningealen Fibroblastenkultur von humanen post mortem Leptomeningen in Verbindung mit einer Gehirn Spende gesammelt abzuleiten. Es gibt sehr wenige Beschreibungen von Protokollen Zellkulturen aus postmortalen menschlichen Material abzuleiten. Zwei Studien beschreiben Haut abgeleiteten Fibroblastkulturen 7, 8, 9, eine Studie beschreibt dura Proben <sup clas...

Meninges bestehen aus drei Membranen, die das Gehirn zu schützen: Dura mater, Arachnoidea und Pia mater. In jüngerer Zeit wurde erkannt , dass Meningen eine wichtige Rolle auch in der Entwicklung des Gehirns und des Gehirns Homöostase 1. Meningen aus mesenchymalen und Neuralleiste abgeleiteten Zellen abgeleitet und interessanterweise , es , dass Vorläuferzellen in Meningen wurden , können in verursachen Neuronen in vitro und vivo nach der Transplantation 2, 3, 4 wohnhaft gezeigt. Meningen Kulturen wurden auch erfolgreich als Feeder - Schichten verwendet werden, wie sie für stromal cell-derived induzierende Aktivität besitzen , die Differenzierung von embryonalen Stammzellen in dopaminerge Neuronen 5. Außerdem haben Leptomeningen das Potential direkt differenzieren sich in Neuronen, Astrozyten und Oligodendrozyten unter ischämischen Bedingungen 6. Aus diesem Protokoll werden menschliche Obduktion Hirnhaut Proben aus der Arachnoidea und Pia mater gesammelt, kollektiv die Leptomeningen und werden als Teil eines menschlichen Gehirns Spende für wissenschaftliche Zwecke beschafft. Sezieren des Gehirns wird in 24 h des Todes durchgeführt und die Probe wird in Leptomeningen Kaltwachstumsmedien für die Weiterverarbeitung in der nächsten 6-8 h angeordnet, wie hier in diesem Protokoll veranschaulicht. Dieses Protokoll beschreibt die Präparation und Herstellung von Human Meningen Proben für die Entwicklung der Patienten primären Leptomeningen Zellkultur. Das Gewebe wird zugeschnitten 25-30 Stücke von ca. 3 mm x 3 mm Quadraten. Drei Stücke sind in jedem 6-Well-Gelatine beschichtete gut und gehalten mit runden Glasplättchen gelegt. Die Meningen Präparation dauert etwa 25-35 min. Die größte Herausforderung bei dieser Kultur ist Sterilität wie das Gehirn Beschaffung, Transport und Präparation werden in der Regel nicht unter sterilen Bedingungen durchgeführt. Therefore, ist es wichtig, die Kulturmedien mit einem Cocktail aus Penicillin, Streptomycin und Amphotericin B und verwenden Multiwellschalen separat Kulturgewebestücke zu ergänzen. Outgrowth meningealer Fibroblasten erfolgt in der Regel innerhalb der ersten Woche. Medium wird alle zwei bis drei Tage gewechselt, bis die Zellen konfluent sind und die Zellen enzymatisch agiert. Die meningeal Fibroblasten sind kryokonservierten bei 1 Million Zellen pro ml / Fläschchen in Kryokonservierung Medien. Mit diesem Protokoll 20-30 Millionen meningealen Fibroblasten können in 6-8 Wochen für die Kryokonservierung ableiten. Downstream Anwendungen dieser meningeal Fibroblasten sind Primärkulturen für die Erforschung von Krankheiten, direkte neuronale Differenzierung oder Ableitung von induzierten pluripotenten Stammzellen, die aus den Leptomeningen für das Verständnis von Krankheitsmechanismen und für die Arzneimittelentwicklung.

<table border="0" cellpadding="0" cellspacing="0" width="1256">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:459px;">Corning Petri dishes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:127px;">351029</td>
			<td style="width:485px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc 6-well plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-mm cover slips</td>
			<td>Fisher Scientific</td>
			<td colspan="2">12-545-83 15CIR-1D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels, sterile blade, No. 15</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved precision tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>22-230490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution</td>
			<td>Fisher Scientific</td>
			<td>SH30264.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 500 mL filter unit</td>
			<td>Fisher Scientific</td>
			<td>430770</td>
			<td>Combine media components and filter.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm2&#160;</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Growth Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hyclone DMEM</td>
			<td>Fisher Scientific</td>
			<td>SH30081.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone FBS</td>
			<td>Fisher Scientific</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B (Yellow Solution/250 &#181;g/mL)</td>
			<td>Fisher Scientific</td>
			<td>BP264520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bambanker Freeze 120 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9582225</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fibronectin Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">8 well chamber slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>1256518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Glycerol&#160;</td>
			<td>BioRad</td>
			<td>9455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% normal goat serum&#160;</td>
			<td>Fisher Scientific</td>
			<td>101098-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Fibronectin antibody [F1]</td>
			<td>Abcam</td>
			<td>ab32419</td>
			<td>1:300 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SERPINH1</td>
			<td>Sigma</td>
			<td>S5950-200ul</td>
			<td>1:250 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SOX2</td>
			<td>Millipore</td>
			<td>MAB4343</td>
			<td>1:100 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td>1:200 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TUJ1</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>1:1000 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td>dilute to a final concentration of 1.0 &#956;g/ml; (blue channel; Ex/Em2 358/461 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Suppliers are suggestions, similar products from alternative vendors can be used as well.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation of leptomeninges explant cultures from postmortem human brain donors

Obwohl große Fortschritte in der klinischen Charakterisierung der Parkinson-Krankheit, einige Studien durchgeführt worden, berichten, dass die Diagnose der Parkinson-Krankheit ist nicht pathologisch bis zu 25% der klinisch diagnostizierten Parkinson-Krankheit bestätigt. Daher ist aus klinisch diagnostizierten Patienten gesammelt Gewebe mit idiopathischen Parkinson-Krankheit eine hohe Rate von Fehldiagnose haben kann; daher in vitro Studien aus solchen Geweben Parkinson-Krankheit zu studieren , wie einem präklinischen Modell zwecklos werden kann. Durch das Sammeln von postmortalen humanen Leptomeningen mit einer bestätigten neuropathologischen Diagnose der Parkinson-Krankheit und gekennzeichnet durch nigrostriatalen Zellverlust und die intrazelluläre Proteineinschlüsse genannt Lewy - Körperchen, kann man sicher sein , dass klinisch beobachtet Parkinsonismus nicht von einem anderen zugrundeliegenden Krankheitsprozess verursacht wird (zB Tumor, Arteriosklerose). Dieses Protokoll presents die Präparation und Herstellung von postmortalen menschlichen Leptomeningen zur Ableitung eines meningeal Fibroblasten-Kultur. Dieses Verfahren ist robust und besitzt eine hohe Erfolgsquote. Die Herausforderung der Kultur liegt Sterilität als das Gehirn Beschaffungs allgemeinen nicht unter sterilen Bedingungen durchgeführt wird. Daher ist es wichtig, die Kulturmedien mit einem Cocktail aus Penicillin, Streptomycin und Amphotericin B. zu ergänzen Die Ableitung der meningealen Fibroblasten von Autopsie bestätigten Fälle mit Parkinson-Krankheit ist die Grundlage für in vitro - Modellierung von Parkinson-Krankheit. Meningeale Fibroblasten erscheinen 3-9 Tage nach der Probenvorbereitung und etwa 20-30 Millionen Zellen können in 6-8 Wochen kryokonserviert werden. Die meningeal Fibroblasten-Kultur ist homogen und die Zellen exprimieren Fibronektin, eine häufig verwendete Marker Hirnhaut zu identifizieren.

Wenn das Leptomeningen Verarbeitungsprotokoll erfolgreich war, Auswuchs meningealer Fibroblasten beobachtet ersten drei bis neun Tage nach der Sektion, obwohl dies von der Länge der Post-mortem-Intervall für das Gehirn verlassen können. Abbildung 1 zeigt meningeal Fibroblasten - Kulturen von vier verschiedenen Spendern. 1A zeigt eine Leptomeningen Stück von einem Deckglas (dunkel diagonale Linie) und Fibroblasten - Auswuchs um das Gewebe vier Tage na...

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

Die Leptomeningen Explantatkultur Protokoll aus menschlichem post mortem Gehirn ist eine technisch robuste und einfache Art und Weise Fibronektin-positive meningeal Fibroblasten innerhalb von 6-8 Wochen und kryokonserviert werden etwa 20-30 Millionen Zellen abzuleiten.

Ableitung von Leptomeningen Explantation Kulturen von 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 ...

derivation-leptomeninges-explant-cultures-from-postmortem-human-brain

Research

JoVE Journal

Neuroscience

8.2K Aufrufe.  Parkinson’s Institute and Clinical Center.  Die Leptomeningen Explantatkultur Protokoll aus menschlichem post mortem Gehirn ist eine technisch robuste und einfache Art und Weise Fibronektin-positive meningeal Fibroblasten innerhalb von 6-8 Wochen und kryokonserviert werden etwa 20-30 Millionen Zellen abzuleiten. 

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

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC Labeling von Neuronen aus Nager und nicht-menschlichen Primaten Hirnschnitten

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Forschung

Neurowissenschaften

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multi-Elektroden-Array Recordings neuronaler Lawinen in organotypischen Kulturen

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Effiziente Ableitung von humanen neuronalen Vorläuferzellen und Neuronen aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

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).

Ableitung von Enriched Oligodendrozyten Kulturen und Oligodendrozyten / Neuron myelinisierenden Co-Kulturen von Post-Natal Mausgeweben

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.

Ableitung von Gliazellen Eingeschränkte Vorstufen aus E13-Mäusen

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 ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Neural Explantatkulturen aus<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Progenitor-abgeleiteten Oligodendrozyten Culture System von Human Fetal Gehirn

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Feeder freie Ableitung von Neural Crest Vorläuferzellen aus menschlichen pluripotenten Stammzellen

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

Hochauflösende Strukturkernspintomographie des menschlichen Subcortex<I&gt; In-vivo-</I&gt; Und Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Isolierung der zerebralen Kapillaren aus frischem menschlichen Hirngewebe

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...