Cette étude a été appuyée par la National Natural Science Foundation of China (Subvention no 81673676) et le Dongguan Science and Technology Bureau (Subvention no 2019622101002). Les auteurs remercient le Dr Maryrose Sullivan (professeure adjointe en chirurgie, Harvard Medical School) pour ses consultations techniques.

Tous les protocoles expérimentaux et les procédures animales étaient conformes aux principes éthiques du Conseil national de recherches du Canada.
1. Préparation des matériaux
<ol><li>Stériliser tous les instruments et ddH 2 Oà l’aided’un autoclave avant d’effectuer l’expérience. Les instruments comprennent, sans s’y limiter, les ciseaux chirurgicaux, les ciseaux ophtalmiques, les forceps, le séparateur de noyau de cuillères, les plats en verre (60-100 mm de diamètre) et les disjoncteurs de verre.</li>
	<li>Préparer la solution Krebs comme suit (Tableau 1):Dissoudre tous les produits chimiques avec ddH2O avant utilisation. Dissoudre le CaCI2 séparément et ajouter lentement dans la solution mixte tout en remuant pour éviter les sédiments.</li>
	<li>Préparer le média Rinse, qui se compose de supports F12 avec 10% de sérum bovin fœtal (FBS) et 1% d’antibiotiques/antimycotiques (100x). Ajouter 5 mL de FBS et 0,5 mL d’antibiotique/antimycotique à 44,5 mL de supports F12.</li>
	<li>Préparer les neurones médias A, qui comprend des médias neurobasaux A avec 2% B-27, 1% FBS, 1% L-glutamine, 1% antibiotique / antimycotique (100x), et 0,1% glia-dérivé facteur neurotrophique (GDNF). Ajouter 200 μL de B-27, 100 μL de FBS, 100 μL de L-glutamine, 100 μL d’antibiotiques/antimycotiques (100x) et 10 μL de GDNF (10 μg/mL) à 9,5 mL de média neurobasal A. REMARQUE : Préparez les neurones media A dans la semaine qui a après l’utilisation pour assurer la fraîcheur du B-27, de la L-glutamine et du GDNF.</li>
	<li>Préparez les neurones médias B en utilisant la même procédure que la préparation des neurones médias A, mais sans ajouter FBS.</li>
	<li>Préparez la solution de digestion 1 en dissolvant 20 mg de collagène de type II, 6 mg d’albumine de sérum bovin et 200 μL d’antibiotique/antimycotique (100x) dans 10 mL de solution Krebs stable en oxygène.</li>
	<li>Préparez la solution de digestion 2 en diluant 1 mL de trypsine de 0,25 % avec 4 mL de solution de sel équilibrée de Hank.</li>
	<li>Préparer une assiette avec des coverslips enduits.<ol>
<li>Utilisez des forceps dans un banc d’écoulement laminaire lorsque vous placez des couvertures en verre dans une plaque de culture de 48 puits.</li>
		<li>Diluer la poly-D-lysine avec le ddH2O à une concentration de 0,1 mg/mL sous forme de bouillon de poly-D-lysine. Conserver le bouillon à -20 °C et décongeler avant utilisation.</li>
		<li>Ajouter 40 μL de bouillon de poly-D-lysine sur chaque coverslip, et incuber la solution à température ambiante pendant 10 min. REMARQUE : Pour différentes plaques avec différentes zones basales, réglez la concentration à 4 μg/cm2. Par exemple, si la surface basale d’un puits d’une plaque de 24 puits est de 2 cm2,alors nous devrions transférer 80 μL de bouillon de poly-D-lysine dans un puits. Chaque cm2 contiendrait 4 μg de poly-D-lysine.</li>
		<li>Retirer la poly-D-lysine dans la plaque de 48 puits et rincer les couvre-plaques avec le ddH2O trois fois.</li>
		<li>Séchez la plaque dans un capot d’écoulement laminaire pendant au moins 30 minutes pour vous assurer que la plaque est anhydre.</li>
		<li>Conserver l’assiette à 4 °C avant de laminer le revêtement pendant 1 jour au plus. Le stockage de la plaque à −20 °C est préférable pour un stockage à long terme.</li>
		<li>Décongeler la laminine à 4 °C. Diluer la laminine avec ddH2O à une concentration de 50 μg/mL comme stock de laminine. Conserver le bouillon à -20 °C et décongeler à 4 °C avant utilisation.</li>
		<li>Utilisez une pipette pour transférer 100 μL de laminine diluée vers le haut de chaque coverslip et incuber laminine à 4 °C pendant 1 h. REMARQUE : Pour différentes plaques avec différentes zones basales, réglez la concentration à 5 μg/cm2. Par exemple, si la surface basale d’un puits d’une plaque de 24 puits est de 2 cm2,puis transférer 200 μL de laminine diluée dans un puits. Chaque cm2 contiendrait 5 μg de laminine.</li>
		<li>Retirez la solution laminine et rincez les coverslips avec ddH2O une fois. Effectuez cette opération sur le bord du coverslip pour éviter le grattage. REMARQUE : Les couvercles enduits peuvent être conservés à 4 °C pendant 2 semaines tout au plus.</li>
	</ol>
</li>
</ol>2. Récolte de vessie
<ol><li>Obtenez des rats Sprague-Dawley âgés de cinq semaines.</li>
	<li>Infuser le carbogen (95% d’oxygène, 5% de CO2)dans la solution Krebs pendant au moins 30 min dans un bain de glace pour atteindre un état d’oxygène stable et un niveau de pH.</li>
	<li>Après l’euthanasie par dislocation cervicale, faire tremper les rats dans 75% d’éthanol pendant 30 s pour la stérilisation.</li>
	<li>Placez les rats sur une serviette chirurgicale stérilisée et exposez leur abdomen. Ouvrez la cavité abdominale, et révélez la vessie avec un ensemble de ciseaux et de forceps.</li>
	<li>Soulevez doucement la vessie et coupez la vessie du col de la vessie avec un autre ensemble de ciseaux et de forceps pour éviter la contamination croisée. Placez rapidement la vessie dans la solution Krebs froide et stable en oxygène pour améliorer la survie des cellules. REMARQUE : Une fois que la vessie est enlevée, effectuez rapidement les opérations suivantes pour améliorer la perspective de survie des neurones.</li>
	<li>Ajouter la solution Krebs dans trois plats en verre et des disjoncteurs en verre.</li>
	<li>Préparez des plats en verre et des disjoncteurs en verre avec la solution Krebs dans un bain de glace pour le précooling.</li>
	<li>Marquez ces conteneurs avec les numéros 1-3 en conséquence pour éviter toute confusion.</li>
	<li>Paire de chaque plat en verre avec des forceps et un diviseur de noyau de cuillères.</li>
	<li>Dans le plat en verre 1, ouvrir la vessie avec des ciseaux ophtalmiques, et la déplier avec des forceps et un séparateur de noyau de cuillères.</li>
	<li>Rincer la vessie dans un disjoncteur en verre 1 et la placer dans un plat en verre 2.</li>
	<li>Éliminer la graisse adhérente sur la surface des tissus à l’aide des forceps et des ciseaux ophtalmiques dans le plat en verre 2.</li>
	<li>Rincer la vessie dans un disjoncteur en verre 2 et la placer dans un plat en verre 3.</li>
	<li>Racler délicatement la vessie à l’aide des forceps et du séparateur du noyau des cuillères sur le plat en verre 3 pour enlever les attaches exogènes.</li>
	<li>Rincer la vessie dans le disjoncteur en verre 3, transférer la vessie dans un tube de centrifugeuse de 15 mL avec 14 mL de solution krebs froide, et faire tourner l’échantillon pendant 1 min à 356 x g et 4 °C.</li>
	<li>Répétez l’étape précédente deux fois dans deux autres tubes avec la solution Krebs pour réduire la contamination.</li>
</ol>3. Digestion de la vessie en deux étapes
<ol><li>Transférer la vessie du tube de centrifugeuse dans un flacon de 2 mL contenant 1 mL de solution de digestion 1. Utilisez des ciseaux ophtalmiques pour couper la vessie en petits morceaux (plus petits que 1 mm) en solution.</li>
	<li>Mélanger la solution vésicale avec 9 mL de solution de digestion 1 dans un plat stérile de culture cellulaire (100 mm de diamètre). Effectuez la digestion de l’étape 1 dans un incubateur secouant pendant 1 h sous 5 % de CO2,37 °C et 200 rpm.</li>
	<li>Après l’étape 1 de la digestion, centrifuger la solution à 356 x g à 4 °C pendant 8 min.</li>
	<li>Placer la solution de digestion 2 dans un bain d’eau de 37 °C pour le préchauffement.</li>
	<li>Après centrifugation, enlever le supernatant qui contient la solution de digestion 1, et récolter les sédiments cellulaires. Un peu de liquide restant est autorisé. L’enlèvement complet de la solution peut favoriser la perte de cellules.</li>
	<li>Mélanger les sédiments cellulaires avec la solution de digestion chaude 2 dans un tube de centrifugeuse de 15 mL, et secouer le mélange tout en digérant dans un bain d’eau de 37 °C pendant 5 min. Ne dépassez pas 7 min de digestion de l’étape 2 ou les neurones périront.</li>
	<li>Après la digestion, désactivez immédiatement la trypsine dans le mélange avec 10 mL de rinçage à froid. REMARQUE : Effectuez les étapes suivantes à 0 °C-4 °C. Un bain de glace pourrait fournir une telle condition.</li>
	<li>Récolter les sédiments cellulaires après centrifugation à 356 x g à 4 °C pendant 8 min. Enlevez autant de médias que possible parce que la trypsine restante est nocive pour la croissance cellulaire.</li>
	<li>Resuspendez les sédiments avec 3 mL de neurones en douceur. Assurez-vous que les bulles d’air ne sont pas générées dans la solution contenant des cellules pour un taux de survie élevé.</li>
	<li>Filtrer le mélange à travers une passoire cellulaire de 70 μm dans un tube de centrifugeuse de 50 mL.</li>
	<li>Garder le filtrate sur un shaker à 30 rpm dans un bain de glace pendant 30 min. Cette étape n’est pas nécessaire mais recommandée.</li>
	<li>Recueillir les cellules par centrifugation à 356 x g à 4 °C pendant 8 min, et resuspendre doucement les granulés cellulaires dans 1 mL de neurone média A.</li>
	<li>Ajouter 500 μL de mélange cellulaire dans chaque puits de la plaque préparée de 48 puits.</li>
	<li>Cellules de culture dans un incubateur à 37 °C et 5% CO2.</li>
	<li>Remplacez tous les médias par des neurones média B en 1 h pour fournir une culture sans sérum.</li>
	<li>Changez la moitié du neurone média B tous les 3 jours. REMARQUE : Les neurones sont prêts pour des expériences immunocytochimiques après 5-7 jours de culture.</li>
</ol>

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Les auteurs ne déclarent aucun conflit d’intérêts majeur.

Préparation des plaques L’utilisation de couvercles de verre dans des plaques de culture de 6, 12 ou 48 puits pour des expériences d’imagerie immunofluorescente ou calcique est une opération économique et économe en échantillons. Les cellules se développent bien dans les plaques sans coverslips pendant la préparation des cultures cellulaires primaires. Par conséquent, les coverslips sont dispensables dans des expériences, telles que la tache occidentale ou la réaction en chaîne de poly...

L’appareil urinaire inférieur a deux fonctions principales : stockage périodique d’urine et micturition1. Le système nerveux inférieur des voies urinaires (LUTNS) contrôle ces fonctions et est délicat et sensible à de nombreuses neuropathies, qui peuvent être innées (porphyrie), acquises (maladie de Lyme), secondaires aux états pathologiques (cystopathie diabétique), médicament induit (cystite hémorragique), chirurgie causée (résection abdominopéine), ou blessures causées (lésions traumatiques de la moelleépinière) 2,3,4,5,6,7. Dans les études physiologiques/pathologiques, les expériences in vivo et in vitro sont tout aussi importantes. Alors que la recherche in vivo sur LUTNS a été menée à des niveaux organiques, cellulaires et moléculaires pendant un certain temps, la recherche in vitro sur les neurones primaires de la vessie urinaire est presque inexistante8,9. Bien que la présente étude soit limitée, nous espérons être les pionniers de la recherche dans ce domaine afin que d’autres chercheurs puissent l’améliorer. De cette façon, cette co-culture peut mener à une compréhension cellulaire du dysfonctionnement physiologique dans les phénotypes, tels que le dysfonctionnement de neurone de réservoir souple.
Contrairement aux muscles entériques avec une directionnalité claire des cellules musculaires en couches discrètes, les muscles de la vessie ne sont pas organisés10. Par conséquent, au lieu d’éplucher la couche externe de la vessie, cette méthode propose de digérer toute la vessie pour réduire la difficulté d’opération et raccourcir le temps de prétraitement pour un taux élevé de survie cellulaire.
En suivant cette méthode, nous pouvons obtenir une culture mixte des neurones et d’autres cellules. Les autres cellules sont indispensables car leur présence imite un environnement in vivo11. En outre, ces cellules fournissent les substances qui ne sont pas disponibles dans le milieu.
Cette méthode implique deux étapes pour la digestion. Tout d’abord, collagène de type II est utilisé pour hydrolyser le collagène, suivie par la trypsine, pour dissocier le tissu encellules 10. De cette façon, les tissus vésicaux sont dispersés en cellules individuelles, puis se développent relativement indépendamment. Lorsque la culture des neurones mûrit, les neurones peuvent être utilisés pour l’imagerie ou des tests fonctionnels.

<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

Les voies urinaires inférieures ont deux fonctions principales, à savoir le stockage périodique de l’urine et la micturition; ces fonctions sont 20 grâce à la neurorégulation centrale et périphérique. Bien que des recherches approfondies sur le système nerveux des voies urinaires inférieures aient été menées, la plupart des études se sont concentrées sur la culture primaire. Ce protocole introduit une méthode pour l’isolement et la culture des neurones vésicaux et des gliales des rats Sprague-Dawley. Dans cette méthode, les neurones et les gliales ont été incubés dans un incubateur de 37 °C, 5 % de CO2 pendant 5 à 7 jours. En conséquence, ils sont devenus des formes matures adaptées aux expériences ultérieures connexes d’immunofluorescence. Des cellules ont été morphologiquement observées utilisant un microscope optique. Les neurones, les vésicules synaptiques et les gliales ont été identifiés par β-III-tubulin et MAP-2, Synapsin-1 et GFAP colorant, respectivement. Pendant ce temps, l’immunocytochimie a été exécutée sur plusieurs protéines neurotransmetteur-connexes, telles que l’acétyltransferase de choline, DYNLL2, et SLC17A9.

Dans le processus de la culture cellulaire primaire, les cellules acquises étaient rondes avec des limites claires et claires avant l’état ci-joint. Au fur et à mesure que les neurones grandissaient, les dendrites et les axones ont commencé à être distincts. Après 5-7 jours de culture, les neurones ont atteint une forme mûre avec de longues projections, qui étaient idéales pour l’imagerie ou les études de fonction. Bien que la plupart des impuretés et des débris cellulaires aient pu être enlevés en rai...

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

Ce protocole tente d’établir un protocole reproductible pour les neurones primaires et l’isolement glia de la vessie de rat pour d’autres expériences cellulaires.

Isolement et culture des neurones primaires et glia de la vessie urinaire adulte de rat

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 vues.  Guangzhou University of Chinese Medicine. Ce protocole tente d’établir un protocole reproductible pour les neurones primaires et l’isolement glia de la vessie de rat pour d’autres expériences cellulaires.

Vidéo: 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.

Nucléofection et de la culture primaire de neurones embryonnaires de souris hippocampe et cortex

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

Recherche

Neurosciences

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.

Didermique co-culture de neurones de rat corticale primaire et des cellules gliales

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.

L&#39;isolement et la culture de neurones de l&#39;hippocampe de souris pendant la grossesse

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.

Une<em&gt; In-vitro</em&gt; Préparation des neurones entériques isolés et cellules gliales du Plexus myentérique de la souris adulte

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.

Culture primaire de souris dopaminergiques neurones

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.

Isolement et culture de dissocié neurones sensoriels De embryons de poulet

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.

Isolement, Culture et maintenance à long terme des mésencéphalique primaire dopaminergiques neurones à partir de cerveaux embryonnaires de rongeurs

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.

Isolement et culture des adultes cellules souches neurales de la Subcallosal Zone Souris

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.

L&#39;isolement et la culture des neurones de la moelle épinière chez les souris néonatales

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.

Culture primaire des neurones isolés du cervelet de souris embryonnaire

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 ...

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Isolement et culture des neurones primaires et glia de la vessie urinaire adulte de rat