Este estudio fue apoyado por la Fundación Nacional de Ciencias Naturales de China (Subvención Nº 81673676) y la Oficina de Ciencia y Tecnología de Dongguan (Subvención Nº 2019622101002). Los autores agradecen a la Dra. Maryrose Sullivan (Profesora Asistente de Cirugía de la Escuela de Medicina de Harvard) por la consultoría técnica.

Todos los protocolos experimentales y procedimientos con animales cumplieron con las directrices de principios éticos del Consejo Nacional de Investigación.
1. Preparación de materiales
<ol><li>Esterilizar todos los instrumentos y ddH2O usando un autoclave antes de realizar el experimento. Los instrumentos incluyen, entre otros, tijeras quirúrgicas, tijeras oftálmicas, fórceps, divisor de núcleo de cucharas, platos de vidrio (60-100 mm de diámetro) y rompeolas de vidrio.</li>
	<li>Preparar la solución Krebs de la siguiente manera (Tabla 1): Disolver todos los productos químicos junto con ddH2O antes de su uso. Disolver CaCI2 por separado y añadir lentamente en la solución mixta mientras se agita para evitar sedimentos.</li>
	<li>Prepare el medio Rinse, que consiste en medios de F12 con 10% suero bovino fetal (FBS) y 1% antibiótico/antimicótico (100x). Añadir 5 ml de FBS y 0,5 ml de antibióticos/antimicóticos a 44,5 ml de medios F12.</li>
	<li>Preparar medios neuron A, que comprende medios neurobasales A con 2% B-27, 1% FBS, 1% L-glutamina, 1% antibiótico/antimicótico (100x), y 0.1% factor neurotrófico derivado de glia (GDNF). Añadir 200 μL de B-27, 100 μL de FBS, 100 μL de L-glutamina, 100 μL de antibiótico/antimicótico (100x) y 10 μL de GDNF (10 μg/ml) a 9,5 ml de media neurobasal A. NOTA: Preparar medios de neuronas A dentro de una semana de uso para garantizar la frescura de B-27, L-glutamina y GDNF.</li>
	<li>Preparar los medios de neuronas B utilizando el mismo procedimiento que la preparación de los medios de neuronas A, pero sin añadir FBS.</li>
	<li>Preparar la solución de digestión 1 disolviendo 20 mg de colágeno tipo II, 6 mg de albúmina sérica bovina y 200 μL de antibiótico/antimicótico (100x) en 10 ml de solución Krebs estable con oxígeno.</li>
	<li>Preparar la solución de digestión 2 diluyendo 1 ml de 0,25% de trippsina con 4 ml de solución de sal equilibrada de Hank.</li>
	<li>Prepare un plato con fundas recubiertas.<ol>
<li>Utilice fórceps en un banco de flujo laminar al colocar tapas de vidrio en una placa de cultivo de 48 pozos.</li>
		<li>Diluir la poli-D-lisina con ddH2O a una concentración de 0,1 mg/ml como poli-D-lisina. Almacene el stock a –20 °C y descongele antes de usarlo.</li>
		<li>Añadir 40 μL de poli-D-lisina en la parte superior de cada tapacubra, e incubar la solución a temperatura ambiente durante 10 minutos. NOTA: Para diferentes placas con diferentes áreas basales, ajuste la concentración a 4 μg/cm2. Por ejemplo, si el área basal de un pozo de una placa de 24 pozos es de 2 cm2,entonces debemos transferir 80 μL de poli-D-lisina en un pozo. Cada cm2 contendría 4 μg de poliD-lisina.</li>
		<li>Retire la poliD-lisina en la placa de 48 pozos y enjuague los cubrebotas con ddH2O tres veces.</li>
		<li>Seque la placa en una campana de flujo laminar durante al menos 30 minutos para asegurarse de que la placa es anhidra.</li>
		<li>Guarde la placa a 4 °C antes del recubrimiento de laminin durante 1 día como máximo. Almacenar la placa a −20 °C es preferible para el almacenamiento a largo plazo.</li>
		<li>Descongelar la laminin a 4 °C. Diluir la laminina con ddH2O a una concentración de 50 μg/mL como caldo de laminina. Guarde el caldo a –20 °C y descongele a 4 °C antes de usarlo.</li>
		<li>Utilice una pipeta para transferir 100 μL de lamina diluida a la parte superior de cada tubo de cubierta e incubar la laminin a 4 °C durante 1 h. NOTA: Para diferentes placas con diferentes áreas basales, ajuste la concentración a 5 μg/cm2. Por ejemplo, si el área basal de un pozo de una placa de 24 pozos es de 2 cm2,luego transfiera 200 μL de laminin diluida en un pozo. Cada cm2 contendría 5 μg de laminin.</li>
		<li>Retire la solución de laminina y enjuague los puntos de cubierta con ddH2O una vez. Realice esta operación en el borde de la cubierta para evitar el raspado. NOTA: Las fundas recubiertas se pueden almacenar a 4 °C durante 2 semanas como máximo.</li>
	</ol>
</li>
</ol>2. Cosecha de vejiga
<ol><li>Consigue ratas Sprague-Dawley de cinco semanas de edad.</li>
	<li>Infundir carburógeno (95% oxígeno, 5% CO2)en la solución Krebs durante al menos 30 minutos en un baño de hielo para alcanzar un estado estable de oxígeno y nivel de pH.</li>
	<li>Después de la eutanasia a través de la dislocación cervical, remoje las ratas en un 75% de etanol durante 30 s para la esterilización.</li>
	<li>Coloque ratas en una toalla quirúrgica esterilizada y exponga su abdomen. Abra la cavidad abdominal y revele la vejiga con un conjunto de tijeras y fórceps.</li>
	<li>Levante la vejiga suavemente y corte la vejiga del cuello de la vejiga con otro conjunto de tijeras y fórceps para evitar la contaminación cruzada. Coloque la vejiga rápidamente en la solución krebs estable en oxígeno frío para mejorar la supervivencia celular. NOTA: Una vez extirpada la vejiga, realice las siguientes operaciones rápidamente para mejorar la perspectiva de supervivencia de las neuronas.</li>
	<li>Agregue la solución Krebs en tres platos de vidrio y rompedores de vidrio.</li>
	<li>Prepare platos de vidrio y vasos con solución Krebs en un baño de hielo para precooling.</li>
	<li>Marque estos contenedores con los números 1–3 correspondientemente para evitar confusiones.</li>
	<li>Empareje cada plato de vidrio con fórceps y un divisor de núcleo de cucharas.</li>
	<li>En el plato de vidrio 1, abre la vejiga con tijeras oftálmicas y despliegala con fórceps y un divisor de núcleo de cucharas.</li>
	<li>Enjuague la vejiga en el interruptor de vidrio 1 y colóquela en el plato de vidrio 2.</li>
	<li>Elimine la grasa adherente en la superficie del tejido utilizando las fórceps y tijeras oftálmicas en la platina de vidrio 2.</li>
	<li>Enjuague la vejiga en el interruptor de vidrio 2 y colóquela en el plato de vidrio 3.</li>
	<li>Raspar suavemente la vejiga usando las fórceps y el divisor del núcleo de cucharas en la platillo de vidrio 3 para eliminar los accesorios exógenos.</li>
	<li>Enjuague la vejiga en el interruptor de vidrio 3, transfiera la vejiga a un tubo centrífuga de 15 ml con 14 ml de solución de Krebs frías y gire la muestra durante 1 min a 356 x g y 4 °C.</li>
	<li>Repita el paso anterior dos veces en otros dos tubos con la solución Krebs para reducir la contaminación.</li>
</ol>3. Digestión de la vejiga de dos pasos
<ol><li>Transfiera la vejiga del tubo centrífuga a un vial de 2 ml que contenga 1 ml de solución de digestión 1. Utilice tijeras oftálmicas para cortar la vejiga en trozos pequeños (menos de 1 mm) en solución.</li>
	<li>Mezcle la solución vesical con 9 ml de solución de digestión 1 en un plato de cultivo celular estéril (100 mm de diámetro). Realice la digestión del paso 1 en una incubadora de agitación durante 1 h bajo 5% CO2,37 °C y 200 rpm.</li>
	<li>Después del paso 1 de digestión, centrífuga la solución a 356 x g a 4 °C durante 8 min.</li>
	<li>Coloque la solución de digestión 2 en un baño de agua a 37 °C para el precalentamiento.</li>
	<li>Después de la centrifugación, retire el sobrenadante que contiene la solución de digestión 1, y coseche el sedimento celular. Se permite el resto del líquido. La eliminación completa de la solución puede promover la pérdida celular.</li>
	<li>Mezcle el sedimento celular con la solución de digestión caliente 2 en un tubo centrífuga de 15 ml y agite la mezcla mientras digiere en un baño de agua de 37 °C durante 5 minutos. No exceda 7 minutos de digestión del paso 2 o las neuronas perecerán.</li>
	<li>Después de la digestión, desactive inmediatamente la triptina en la mezcla con 10 ml de medios de enjuague en frío. NOTA: Realice los siguientes pasos a 0 °C–4 ° C. Un baño de hielo podría proporcionar tal condición.</li>
	<li>Coseche el sedimento celular después de la centrifugación a 356 x g a 4 °C durante 8 min. Retire tantos medios como sea posible porque la trypsina restante es perjudicial para el crecimiento celular.</li>
	<li>Sedimentos resuspend con 3 ml de media neurona suavemente. Asegúrese de que las burbujas de aire no se generan en la solución que contiene células para una alta tasa de supervivencia.</li>
	<li>Filtre el medio de mezcla a través de un colador de células de 70 μm en un tubo centrífuga de 50 ml.</li>
	<li>Mantenga el filtrador en una coctelera a 30 rpm en un baño de hielo durante 30 minutos. Este paso no es necesario, pero se recomienda.</li>
	<li>Recoger las células por centrifugación a 356 x g a 4 °C durante 8 min, y resuspend suavemente los pellets celulares en 1 ml de media de neurona A.</li>
	<li>Agregue 500 μL de mezcla celular en cada pozo de la placa de 48 pozos preparada.</li>
	<li>Células de cultivo en una incubadora a 37 °C y 5% CO2.</li>
	<li>Reemplace todos los medios por los medios de neuronas B en 1 h para proporcionar una cultura libre de suero.</li>
	<li>Cambie la mitad de los medios de neuronas B cada 3 días. NOTA: Las neuronas están listas para experimentos inmunocitoquímicos después de 5-7 días de cultivo.</li>
</ol>

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Los autores no declaran ningún conflicto de intereses importante.

Preparación de placas El uso de manchas de vidrio en placas de cultivo de 6, 12 o 48 pozos para experimentos de imágenes inmunofluorescentes o de calcio es una operación económica y que ahorra muestras. Las células crecen bien en placas sin manchas durante la preparación de cultivos celulares primarios. Por lo tanto, los coverslips son prescindibles en experimentos, como la blot occidental o la reacción en cadena de la polimerasa. Además, el recubrimiento es un paso necesario antes de atorniga...

Las vías urinarias inferiores tienen dos funciones principales: almacenamiento periódico de orina y micturición1. El sistema nervioso del tracto urinario inferior (LUTNS) controla estas funciones y es delicado y susceptible a muchas neuropatías, que pueden ser innatas (porfiria), adquiridas (enfermedad de Lyme), secundarias a estados de enfermedad (cistopatía diabética), inducida por fármacos (cistitis hemorrágica), cirugía causada (resección abjope) o lesión causada (lesión traumática de la médula espinal)2,3,4,5,6,7. En estudios fisiológicos/patológicos, los experimentos in vivo e in vitro son igualmente importantes. Mientras que la investigación in vivo sobre LUTNS se ha llevado a cabo a niveles de órganos, celulares y moleculares durante algún tiempo, la investigación in vitro sobre las neuronas primarias de la vejiga urinaria es casi inexistente8,9. Aunque el presente estudio es limitado, esperamos ser pioneros en la investigación en esta área para que otros investigadores puedan mejorarla. De esta manera, este co-cultivo puede conducir a una comprensión celular de la disfunción fisiológica en fenotipos, como la disfunción de las neuronas de la vejiga.
A diferencia de los músculos entéricos con una dirección clara de las células musculares en capas discretas, los músculos de la vejiga están desorganizados10. Por lo tanto, en lugar de despegar la capa externa de la vejiga, este método propone digerir toda la vejiga para reducir la dificultad de operación y acortar el tiempo de preprocesamiento para una alta tasa de supervivencia celular.
Siguiendo este método, podemos obtener un cultivo mixto de neuronas y otras células. Las otras células son indispensables porque su presencia imita un entorno in vivo11. Además, estas células proporcionan las sustancias que no están disponibles en el medio.
Este método implica dos pasos para la digestión. En primer lugar, la colágeno tipo II se utiliza para hidrolizando colágeno, seguido de trippsina, para disociar el tejido en las células10. De esta manera, los tejidos de la vejiga se dispersan en células individuales y luego crecen relativamente independientes. Cuando el cultivo de las neuronas madura, las neuronas se pueden utilizar para imágenes o ensayos funcionales.

<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

Las vías urinarias inferiores tienen dos funciones principales, a saber, el almacenamiento periódico de orina y la micturición; estas funciones se median a través de la neuroregulación central y periférica. Aunque se ha realizado una extensa investigación sobre el sistema nervioso del tracto urinario inferior, la mayoría de los estudios se han centrado en el cultivo primario. Este protocolo introduce un método para el aislamiento y cultivo de las neuronas de la vejiga y la glia de las ratas Sprague-Dawley. En este método, las neuronas y la glia fueron incubadas en una incubadora de 37 °C, 5% CO2 durante 5-7 días. Como resultado, se convirtieron en formas maduras adecuadas para experimentos de inmunofluorescencia posteriores relacionados. Las células fueron observadas morfológicamente usando un microscopio óptico. Las neuronas, las vesículas sinápticas y la glia fueron identificadas por β-III-tubulina y MAP-2, Synapsin-1 y manchas GFAP, respectivamente. Mientras tanto, la inmunocitoquímica se realizó en varias proteínas relacionadas con el neurotransmisor, como la acetiltransferasa de colina, DYNLL2 y SLC17A9.

En el proceso de cultivo celular primario, las células adquiridas eran redondas con límites brillantes y claros antes del estado adjunto. A medida que las neuronas crecían, los dendritas y los axones comenzaron a ser distintos. Después de 5-7 días de cultivo, las neuronas alcanzaron una forma madura con proyecciones largas, que eran ideales para estudios de imágenes o funciones. Aunque la mayoría de las impurezas y desechos celulares podrían ser retirados debido al cambio de medios, ciertos residuos unidos al rec...

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

Este protocolo intenta establecer un protocolo repetible para las neuronas primarias y el aislamiento de glia de la vejiga de rata para experimentos celulares posteriores.

Aislamiento y cultura de las neuronas primarias y Glia de la vejiga urinaria de rata adulta

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 vistas.  Guangzhou University of Chinese Medicine. Este protocolo intenta establecer un protocolo repetible para las neuronas primarias y el aislamiento de glia de la vejiga de rata para experimentos celulares posteriores.

Video: 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 y cultivos primarios de neuronas del hipocampo de embriones de ratón y corticales

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

Investigación

Neurociencias

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.

Bilaminar Co-cultivo de neuronas de rata primaria cortical y la glía

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.

Aislamiento y cultivo de neuronas del hipocampo de ratones prenatales

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.

Un<em&gt; In-vitro</em&gt; Preparación de las neuronas entéricas aisladas y células gliales del plexo mientérico del ratón adulto

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.

Cultivo primario de ratón neuronas dopaminérgicas

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.

Aislamiento y cultivo de neuronas sensoriales disociadas De embriones de pollo

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.

Aislamiento, cultivo y mantenimiento a largo plazo de Primaria mesencefálico neuronas dopaminérgicas a partir de cerebros de embriones de roedores

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.

Aislamiento y cultivo de células madre adultas neural de la Zona subcallosal Ratón

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.

Aislamiento y Cultura de Neuronas de la Médula Espinal de Ratones Neonatales

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.

Cultivo primario de neuronas aisladas del cerebelo del ratón embrionario

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 ...

Methods Article

Aislamiento y cultura de las neuronas primarias y Glia de la vejiga urinaria de rata adulta