Diese Studie wurde von der National Natural Science Foundation of China (Grant-Nr. 81673676) und dem Dongguan Science and Technology Bureau (Grant-Nr. 2019622101002) unterstützt. Die Autoren danken Dr. Maryrose Sullivan (Assistant Professor in Surgery, Harvard Medical School) für die technische Beratung.

Alle Versuchsprotokolle und Tierverfahren entsprachen den ethischen Grundsätzen des Nationalen Forschungsrates.
1. Herstellung von Materialien
<ol><li>Sterilisieren Sie alle Instrumente und ddH2O mit einem Autoklaven, bevor Sie das Experiment durchführen. Zu den Instrumenten gehören u.a. chirurgische Schere, Augenscheren, Zangen, Löffelkernteiler, Glasschalen (60–100 mm Durchmesser) und Glasbrecher.</li>
	<li>Bereiten Sie die Krebs-Lösung wie folgt vor (Tabelle 1): Alle Chemikalien zusammen mit ddH2O vor gebrauchen auflösen. CaCI2 separat auflösen und unter Rühren langsam in die Mischlösung geben, um Sedimente zu vermeiden.</li>
	<li>Bereiten Sie die Spülmedien vor, die aus F12-Medien mit 10% fetalem Rinderserum (FBS) und 1% Antimykotika (100x) bestehen. Fügen Sie 5 ml FBS und 0,5 ml Antibiotikum/Antimykotik um 44,5 ml F12-Medien hinzu.</li>
	<li>Bereiten Sie neuronen Medien A, die neurobasale A-Medien mit 2% B-27, 1% FBS, 1% L-Glutamin, 1% Antimykotik (100x) und 0,1% Glia-abgeleiteten neurotrophen Faktor (GDNF) umfasst. Fügen Sie 200 l B-27, 100 l FBS, 100 l L L-Glutamin, 100 l Antibiotikum/Antimykotik (100x) und 10 l GDNF (10 g/ml) bis 9,5 ml neurobaales A-Medium hinzu. HINWEIS: Bereiten Sie Neuronenmedien A innerhalb einer Woche nach gebrauch, um die Frische von B-27, L-Glutamin und GDNF zu gewährleisten.</li>
	<li>Bereiten Sie Neuronenmedien B mit dem gleichen Verfahren wie die Vorbereitung von Neuronenmedien A vor, ohne jedoch FBS hinzuzufügen.</li>
	<li>Bereiten Sie die Verdauungslösung 1 vor, indem Sie 20 mg Kollagenase Typ II, 6 mg Rinderserumalbumin und 200 l Antibiotikum/Antimykotik (100x) in 10 ml sauerstoffstabiler Krebslösung auflösen.</li>
	<li>Bereiten Sie die Verdauungslösung 2 vor, indem Sie 1 ml 0,25% Trypsin mit 4 ml der ausgewogenen Salzlösung von Hank verdünnen.</li>
	<li>Bereiten Sie eine Platte mit beschichteten Abdeckungen.<ol>
<li>Verwenden Sie Zangen in einer laminaren Strömungsbank, wenn Sie Glasabdeckungen in eine 48-Well-Kulturplatte legen.</li>
		<li>Poly-D-Lysin mitddH2O auf eine Konzentration von 0,1 mg/ml als Poly-D-Lysin-Bestand verdünnen. Lagern Sie den Lagerbestand bei -20 °C und tauen Sie vor Gebrauch.</li>
		<li>Fügen Sie 40 L Poly-D-Lysin-Lager auf jedem Deckelschlupf hinzu und inkubieren Sie die Lösung bei Raumtemperatur für 10 min. ANMERKUNG: Stellen Sie bei verschiedenen Platten mit unterschiedlichen Basalbereichen die Konzentration auf 4 g/cm2ein. Wenn z.B. die Basalfläche eines Brunnens aus einer 24-Well-Platte 2 cm2beträgt, dann sollten wir 80 l Poly-D-Lysin-Bestand in einen Brunnen übertragen. Jeder cm2 würde 4 g Poly-D-Lysin enthalten.</li>
		<li>Entfernen Sie Poly-D-Lysin in der 48-Well-Platte und spülen Sie Dielipse mit ddH2O dreimal.</li>
		<li>Trocknen Sie die Platte in einer laminaren Durchflusshaube für mindestens 30 min, um sicherzustellen, dass die Platte wasserfrei ist.</li>
		<li>Lagern Sie die Platte bei 4 °C vor der Laminierung höchstens 1 Tag. Die Lagerung der Platte bei 20 °C ist für eine Langzeitlagerung vorzuziehen.</li>
		<li>Laminin bei 4 °C auftauen. Laminin mit ddH2O auf eine Konzentration von 50 g/ml als Lamininvorrat verdünnen. Lagern Sie den Lagerbestand bei -20 °C und tauen Sie vor Gebrauch bei 4 °C auf.</li>
		<li>Verwenden Sie eine Pipette, um 100 l verdünntes Laminin an die Oberseite jedes Deckellipszuglases zu übertragen und Laminin bei 4 °C für 1 h zu inkubieren. ANMERKUNG: Stellen Sie bei verschiedenen Platten mit unterschiedlichen Basalbereichen die Konzentration auf 5 g/cm2ein. Wenn z. B. die Basalfläche eines Brunnens aus einer 24-Well-Platte 2 cm2beträgt, dann 200 l verdünntes Laminin in einen Brunnen geben. Jede cm2 würde 5 g Laminin enthalten.</li>
		<li>Entfernen Sie die Lamininlösung und spülen Sie die Abdeckungen einmal mit ddH2O. Führen Sie diesen Vorgang am Rand des Deckslips aus, um Einschübe zu vermeiden. HINWEIS: Beschichtete Abdeckungen können höchstens 2 Wochen bei 4 °C gelagert werden.</li>
	</ol>
</li>
</ol>2. Blasenernte
<ol><li>Erhalten Sie fünf Wochen alte Sprague-Dawley-Ratten.</li>
	<li>Indercarbogen (95% Sauerstoff, 5%CO2) in die Krebs-Lösung für mindestens 30 min in einem Eisbad, um einen stabilen Sauerstoffzustand und pH-Wert zu erreichen.</li>
	<li>Nach der Euthanasie durch Zervixdislokation die Ratten in 75% Ethanol für 30 s zur Sterilisation einweichen.</li>
	<li>Legen Sie Ratten auf ein sterilisiertes chirurgisches Handtuch und setzen Sie ihren Bauch aus. Öffnen Sie die Bauchhöhle, und offenbaren Sie die Blase mit einer Reihe von Scheren und Zangen.</li>
	<li>Heben Sie die Blase sanft an und schneiden Sie die Blase mit einer weiteren Schere und Zange aus dem Blasenhals, um Kreuzkontaminationen zu vermeiden. Legen Sie die Blase schnell in kalte sauerstoffstabile Krebslösung, um das Zellüberleben zu verbessern. HINWEIS: Sobald die Blase entfernt ist, führen Sie die folgenden Operationen schnell durch, um die Aussicht auf neuronen Überleben zu verbessern.</li>
	<li>Fügen Sie Krebs-Lösung in drei Glasschalen und Glasbrecher.</li>
	<li>Bereiten Sie Glasgeschirr und Glasbrecher mit Krebs-Lösung in einem Eisbad zur Vorkühlung vor.</li>
	<li>Markieren Sie diese Behälter mit den Zahlen 1–3 entsprechend, um Verwechslungen zu vermeiden.</li>
	<li>Kombinieren Sie jede Glasschale mit Zangen und einem Löffel Kernteiler.</li>
	<li>In Glasschale 1 die Blase mit einer Augenschere aufschneiden und mit Zangen und einem Löffelkernteiler aufklappen.</li>
	<li>Spülen Sie die Blase in Glasbrecher 1, und legen Sie es in Glasschale 2.</li>
	<li>Beseitigen Sie anhaftendes Fett auf der Gewebeoberfläche mit den Zangen und der Augenschere in Glasschale 2.</li>
	<li>Spülen Sie die Blase in Glasbrecher 2, und legen Sie es in Glasschale 3.</li>
	<li>Kratzen Sie die Blase vorsichtig mit den Zangen und dem Löffelkernteiler auf Glasschale 3, um exogene Anhänge zu entfernen.</li>
	<li>Spülen Sie die Blase im Glasbrecher 3, übertragen Sie die Blase in ein 15 ml Zentrifugenrohr mit 14 ml kalter Krebslösung und drehen Sie die Probe 1 min bei 356 x g und 4 °C.</li>
	<li>Wiederholen Sie den vorherigen Schritt zweimal in zwei anderen Rohren mit Krebs-Lösung, um die Kontamination zu reduzieren.</li>
</ol>3. Zweistufige Blasenverdauung
<ol><li>Die Blase aus dem Zentrifugenrohr in eine 2 ml Durchstechflasche mit 1 ml Verdauungslösung 1 übertragen. Verwenden Sie eine Augenschere, um die Blase in kleine Stücke (kleiner als 1 mm) in Lösung zu schneiden.</li>
	<li>Mischen Sie die Blasenlösung mit 9 ml Verdauungslösung 1 in einer sterilen Zellkulturschale (100 mm Durchmesser). Schritt 1 Verdauung in einem Schüttelinkubator für 1 h unter 5%CO2,37 °C und 200 U/min durchführen.</li>
	<li>Nach Schritt 1 Vergärung zentrieren Sie die Lösung bei 356 x g bei 4 °C für 8 min.</li>
	<li>Die Verdauungslösung 2 zur Vorwärmung in ein 37 °C-Wasserbad geben.</li>
	<li>Entfernen Sie nach der Zentrifugation den Überstand, der die Verdauungslösung 1 enthält, und ernten Sie das Zellsediment. Etwas Restflüssigkeit ist erlaubt. Vollständige Entfernung der Lösung kann Zellverlust fördern.</li>
	<li>Zellsediment mit warmer Verdauungslösung 2 in einem 15 ml Zentrifugenrohr mischen und die Mischung schütteln, während sie in einem 37 °C-Wasserbad für 5 min verdauen. Nicht mehr als 7 min Schritt 2 Verdauung oder Neuronen gehen unter.</li>
	<li>Nach der Verdauung Sofort Trypsin in der Mischung mit 10 ml Kaltspülmedium deaktivieren. HINWEIS: Führen Sie die folgenden Schritte bei 0 °C–4 °C aus. Ein Eisbad könnte einen solchen Zustand bieten.</li>
	<li>Das Zellsediment nach Zentrifugation bei 356 x g bei 4 °C für 8 min ernten. Entfernen Sie so viele Medien wie möglich, da das verbleibende Trypsin schädlich für das Zellwachstum ist.</li>
	<li>Sediment mit 3 ml Neuronenmedien sanft aufsetzen. Stellen Sie sicher, dass Luftblasen nicht in der Lösung erzeugt werden, die Zellen für eine hohe Überlebensrate enthält.</li>
	<li>Filtern Sie das Mischungsmedium durch ein 70-m-Zellsieb in ein 50 ml Zentrifugenrohr.</li>
	<li>Halten Sie das Filtrat bei 30 Umdrehungen pro Minute in einem Eisbad 30 min auf einem Shaker. Dieser Schritt ist nicht notwendig, wird aber empfohlen.</li>
	<li>Sammeln Sie Zellen durch Zentrifugation bei 356 x g bei 4 °C für 8 min, und setzen Sie die Zellpellets in 1 ml Neuronenmedium A sanft wieder auf.</li>
	<li>Fügen Sie 500 l Zellmischung in jeden Brunnen der vorbereiteten 48-Well-Platte.</li>
	<li>Kulturzellen in einem Inkubator bei 37 °C und 5%CO2.</li>
	<li>Ersetzen Sie alle Medien durch Neuronenmedien B in 1 h, um eine serumfreie Kultur bereitzustellen.</li>
	<li>Ändern Sie alle 3 Tage die Hälfte der Neuronenmedien B. HINWEIS: Neuronen sind bereit für immunzytochemische Experimente nach 5–7 Tagen Kultur.</li>
</ol>

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Die Autoren erklären keinen größeren Interessenkonflikt.

Plattenvorbereitung Die Verwendung von Glasabdeckungen in 6-, 12- oder 48-Well-Kulturplatten für immunfluoreszierende oder Kalzium-Bildgebungsexperimente ist eine wirtschaftliche und probenschonende Operation. Zellen wachsen gut in Platten ohne Decklippen während der Herstellung von primären Zellkulturen. Daher sind Coverlips in Experimenten wie Western Blot oder Polymerase-Kettenreaktion entbehrlich. Darüber hinaus ist die Beschichtung ein notwendiger Schritt vor der Beschichtung von Zellen, mit ...

Der untere Harnweg hat zwei Hauptfunktionen: periodische Urinlagerung und Micturition1. Die unteren Harnwege Nervensystem (LUTNS) steuert diese Funktionen und ist empfindlich und anfällig für viele Neuropathien, die angeboren sein können (Porphyrie), erworben (Lyme-Krankheit), sekundär zu Krankheitszuständen (diabetische Zystopathie), medikamentös induziert (hämorrhagische Zystitis), Chirurgie verursacht (abdominoperinealresektion), oder Verletzungen verursacht (traumatische Rückenmarksverletzung)2,3,4,5,6. In physiologisch-pathologischen Studien sind In-vivo- und In-vitro-Experimente gleichermaßen wichtig. Während in vivo Forschung auf LUTNS wurde auf Organ-, zelluläre, und molekulare Ebene für einige Zeit durchgeführt, In-vitro-Forschung an primären Neuronen aus der Harnblase ist fast nicht existent8,9. Obwohl die vorliegende Studie begrenzt ist, hoffen wir, die Forschung in diesem Bereich voranzutragen, damit andere Forscher sie verbessern können. Auf diese Weise, Diese Co-Kultur kann zu einem zellulären Verständnis der physiologischen Dysfunktion in Phänotypen führen, wie Blase Neuron Dysfunktion.
Im Gegensatz zu enterischen Muskeln mit einer klaren Richtung der Muskelzellen in diskrete Schichten sind die Muskeln der Blase unorganisiert10. Anstatt die äußere Schicht der Blase abzuschälen, schlägt diese Methode daher vor, die gesamte Blase zu verdauen, um die Schwierigkeit des Betriebs zu reduzieren und die Vorverarbeitungszeit für eine hohe Zellüberlebensrate zu verkürzen.
Nach dieser Methode können wir eine gemischte Kultur von Neuronen und anderen Zellen erhalten. Die anderen Zellen sind unentbehrlich, da ihre Anwesenheit eine in vivo Umgebung imitiert11. Darüber hinaus liefern solche Zellen die Substanzen, die im Medium nicht verfügbar sind.
Diese Methode umfasst zwei Schritte für die Verdauung. Zunächst wird Kollagenase Typ II verwendet, um Kollagen zu hydrolysieren, gefolgt von Trypsin, um das Gewebe in Zellen10zu dissoziieren. Auf diese Weise werden Blasengewebe in einzelne Zellen dispergiert und wachsen dann relativ unabhängig. Wenn die Kultur der Neuronen reift, können die Neuronen für bildgebende oder funktionelle Assays verwendet werden.

<table border="0" cellpadding="0" cellspacing="0" style="width:1061px;" width="1061">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.25% trypsin</td>
			<td style="width:279px;">Gibico</td>
			<td style="width:119px;">15050065</td>
			<td style="width:296px;">Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48-well culture plate</td>
			<td>Corning</td>
			<td style="width:119px;">3548</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibiotic/antimycotic</td>
			<td>Gibico</td>
			<td>15240062</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Glial Fibrillary Acidic Protein Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-33673</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27</td>
			<td>Gibico</td>
			<td>17504044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA Fraction V</td>
			<td>Gibico</td>
			<td>332</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Choline Acetyltransferase Antibody</td>
			<td>Abcam</td>
			<td>ab18736</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 Incubator</td>
			<td>Heraeus</td>
			<td>B16UU</td>
			<td>Cells culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type II</td>
			<td>Sigma</td>
			<td>2593923</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">DMEM/F-12</td>
			<td>Gibico</td>
			<td>11330032</td>
			<td>Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DYNLL2 Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-13969</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gibico</td>
			<td>10100147</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>1383</td>
			<td>10 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass breakers</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1101</td>
			<td>50 ml; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>WHB Scientific</td>
			<td>WHB-48-CS</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1177</td>
			<td>100 mm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>BD</td>
			<td>561908</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow bench</td>
			<td>Su Jie Medical Devices</td>
			<td>CB 1400V</td>
			<td>Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Antibody</td>
			<td>Affinity</td>
			<td>AF5156</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murine GDNF</td>
			<td>Peprotech</td>
			<td>AF45044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal-A Medium</td>
			<td>Gibico</td>
			<td>10888022</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophthalmic scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21010</td>
			<td>12.5 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000240</td>
			<td>100-1000 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000267</td>
			<td>10-100 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated centrifuge</td>
			<td>Ping Fan Instrument</td>
			<td>TGL-16A</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Haimen Kylin-Bell Lab Instruments</td>
			<td style="width:119px;">T8-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SLC17A9 Antibody</td>
			<td>MBL International</td>
			<td>BMP079</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spoons nucleus divider</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>YZR030</td>
			<td>12 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substance P Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-58591</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21130</td>
			<td>16 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical towel</td>
			<td>Fu Kang Medical Devices</td>
			<td>5002</td>
			<td>40 x 50 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synapsin-1 Antibody</td>
			<td>CST</td>
			<td>5297T</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubulin beta Antibody(&#946;-III-tubulin)</td>
			<td>Affinity</td>
			<td>AF7011</td>
			<td>ICC</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary neurons and glia from adult rat urinary bladder

Die unteren Harnwege hat zwei Hauptfunktionen, nämlich regelmäßige Urinlagerung und Micturition; diese Funktionen werden durch zentrale und periphere Neuroregulation vermittelt. Obwohl umfangreiche Forschung über die unteren Harnwege Nervensystem durchgeführt wurde, die meisten Studien haben sich auf primärkultur konzentriert. Dieses Protokoll führt eine Methode zur Isolierung und Kultur von Blasenneuronen und Glia von Sprague-Dawley Ratten ein. Bei dieser Methode wurden die Neuronen und Glia in einem 37 °C, 5% CO2-Inkubator für 5–7 Tage inkubiert. Infolgedessen wuchsen sie zu ausgereiften Formen heran, die für verwandte nachfolgende Immunfluoreszenzexperimente geeignet waren. Die Zellen wurden mit einem optischen Mikroskop morphologisch beobachtet. Neuronen, synaptische Vesikel und Glia wurden durch β-III-Tubulin und MAP-2, Synapsin-1 bzw. GFAP-Färbung identifiziert. In der Zwischenzeit wurde die Immunzytochemie an mehreren Neurotransmitter-bezogenen Proteinen durchgeführt, wie Cholin-Acetyltransferase, DYNLL2 und SLC17A9.

Im Prozess der primären Zellkultur waren die erfassten Zellen rund mit hellen und klaren Grenzen vor dem angefügten Zustand. Als die Neuronen wuchsen, begannen Dendriten und Axone zu unterscheiden. Nach 5–7 Tagen Kultur erreichten die Neuronen eine reife Form mit langen Projektionen, die ideal für Bildgebungs- oder Funktionsstudien waren. Obwohl die meisten Verunreinigungen und Zellablagerungen aufgrund wechselnder Medien entfernt werden konnten, waren bestimmte Residuen, die an Poly-D-Lysin- und Lamininbeschichtung...

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Dieses Protokoll versucht, ein wiederholbares Protokoll für primäre Neuronen und Gliaisolation von Rattenblase für weitere zelluläre Experimente zu etablieren.

Isolation und Kultur der primär neuronen und Glia von Adult Ratte Harnblase

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Research

JoVE Journal

Neuroscience

3.8K Aufrufe.  Guangzhou University of Chinese Medicine. Dieses Protokoll versucht, ein wiederholbares Protokoll für primäre Neuronen und Gliaisolation von Rattenblase für weitere zelluläre Experimente zu etablieren.

Serie: Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection und Primärkultur von embryonalen Maus-Hippocampus und kortikalen Neuronen

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Forschung

Neurowissenschaften

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminäre Co-Kultur von primären Ratten-kortikalen Neuronen und Glia

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

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolierung und Kultivierung von Neuronen im Hippocampus von Mäusen Pränatale

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Ein<em&gt; In-vitro</em&gt; Vorbereitung von isolierten Neuronen und Glia Enteric vom Plexus myentericus der erwachsenen Maus

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Primärkultur von Maus dopaminergen Neuronen

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolation und Kultur der Dissoziierte sensorischen Neuronen aus Hühnerembryonen

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolation, Kultur und langfristige Wartung von Primär mesencephalen dopaminergen Neuronen aus embryonalen Nagetier Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolierung und Kultur von adulten neuralen Stammzellen aus der Maus subcallosa Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Wirbelsäule Neuronen Isolierung und Kultur aus neonatalen Mäusen

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Primäre Kultur der Neuronen isoliert von Embryonal Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...