Damos las gracias a Jonna Ellis por su asistencia editorial. El proyecto descrito fue apoyada por el premio número R01MH079751 (IP: F. Peruzzi) del Instituto Nacional de Salud Mental. El contenido es responsabilidad exclusiva de sus autores y no representa necesariamente las opiniones oficiales del Instituto Nacional de Salud Mental o los Institutos Nacionales de Salud.

1. Poly-D-lisina (PDL): Preparación <ol><li> Añadir 5 ml de ddH estéril 2 O a 5 mg de PDL para obtener una solución madre de 1 mg / ml. </li><li> Mezcle la solución stock de pipetear varias veces. </li><li> Usar inmediatamente o almacenar poli-D-lisina solución a 2-8 ° C. </li></ol> 2. Poly-D-lisina (PDL): revestimiento de plástico placas de cultivo de células <ol><li> Diluir la solución madre de PDL con estéril O ddH 2 a la concentración final de 10 g / ml. </li><li> Pipeta suficiente solución en un plato de 60 mm para cubrir el área cultivo de superficie (3 ml para un plato mm 60). </li><li> Roca suavemente para asegurar un recubrimiento de la superficie de cultivo. </li><li> Incubar las placas revestidas a temperatura ambiente (TA) durante la noche. </li><li> Al día siguiente, por lo general el día de la disección, se retira la solución de poli-D-lisina por aspiración y se lava brevemente con 3 ml de ddH estéril O. 2 Repita este paso. Tras el segundo lavado, eliminar el agua completamente por aspiración. </li><li>Las placas pueden ser almacenadas a 4 ° C durante hasta tres semanas. </li></ol> 3. Poly-D-lisina (PDL) y laminina: Preparación y revestimiento de vidrio de dos cámaras de diapositivas <ol><li> Mezcla PDL (1 mg / ml) y laminina (1 mg / ml) en soluciones madre estéril O ddH 2 a la concentración final de 10 y 5 ug / ml, respectivamente. </li><li> Pipeta suficiente solución en los pocillos de un vidrio de dos cámaras de diapositivas para cubrir el área de superficie de cultivo (1 ml para cada pocillo de un vidrio 2 también de dos cámaras de diapositivas). </li><li> Roca suavemente para asegurar un recubrimiento de la superficie de cultivo. </li><li> Incubar las placas revestidas a TA durante la noche. </li><li> Al día siguiente, retire la solución de revestimiento de poli-D-lisina-laminina mediante aspiración y lavar suavemente dos veces con 1 ml de agua estéril ddH 2 O. Tras el segundo lavado, eliminar el agua completamente por aspiración. </li><li> Cámara de diapositivas se puede almacenar a 4 ° C durante hasta tres semanas. </li></ol> Nota: Cualquier diapositiva cámara de vidrio se pueden recubrir following este protocolo. A menudo utilizamos las diapositivas de dos cámaras, ya que cada diapositiva proporciona la configuración del control de la prueba experimental (por ejemplo, no se trata en comparación con tratamiento, en comparación con untransfected transfectadas). 4. Disección neuronal y Cultura <ol><li> Calentar los siguientes reactivos en un baño de agua 37 ° C: <ul type="disc"><li> TrypLE Express en su original botella de 100 ml. </li><li> Neurobasal/B27 medio completo (véase I cuadro). El volumen calentado depende del número de platos para ser chapado (por ejemplo 30 ml de diez placas de 60 mm chapados). </li></ul></li><li> Añadir 3 ml de solución fría de hibernación E para cuatro placas de 60 mm y 13 ml de cultivo a un tubo de 15 ml BD Falcon polipropileno de alta claridad cónica. </li><li> Añadir 25-30 ml de medio de disección en frío (véase el cuadro II, el Dr. Olimpia Meucci, comunicación personal) para cada uno de los tres platos de 100 mm cultura. Estas placas, que contienen un gran volumen de medio, se utiliza para lavar los embriones inmediatamente después de sula eliminación de las bolsas amnióticas (medidas 4.7 y 4.8). </li><li> La eutanasia de una rata E17 cronometrado embarazada de CO 2 de acuerdo con la Política de Servicios de Salud Pública de Cuidado Humano y Uso de Animales de Laboratorio y bajo un cuidado de los animales y el uso institucionalmente aprobó el protocolo. </li><li> Rociar con el abdomen inferior 70% de EtOH y corte medial a través de la piel y los músculos con un par de tijeras que exponen el útero y los embriones. </li><li> Retire todos los fetos y colóquelos en un recipiente estéril de 100 mm plato que contiene un exceso de medio de disección en frío (25-30 ml, véase el paso 4.3). </li><li> Cortar los embriones con un pequeño par de tijeras de saco amniótico y colocarlos en el segundo plato de 100 mm que contenían medio de disección fría. </li><li> Lavar los embriones a temperatura ambiente inclinando suavemente el plato 100-mm durante 5-10 segundos. Luego, transferir los embriones lavados al plato mm tercera 100 que contiene el medio de disección. Dos lavados en medio de un exceso suelen ser suficientes para eliminar todos los rastros de sangre. Sin embargo, si ncopcuando fuere necesario, lavar una vez más con una nueva de 100 mm, plato que contiene 25-30 ml de medio de disección en frío. </li><li> Utilizando una pinza estereomicroscopio y curvos, extraer el cerebro de cada embrión de rata tirando de la piel y el cráneo. Coloque todo el cerebro en uno de los platos de 60 mm (por lo general, con no más de 5 cerebros por plato) con Hibernate fría E. Mantener estas placas de hielo. </li><li> Tome un plato a la vez y, en virtud de un microscopio de disección, separar los hemisferios y aislar a la eliminación de las cortezas cerebrales del cerebro medio y las meninges. </li><li> Opcional: los cerebros de corte a lo largo de la línea media, extraer los hipocampos, y siga el siguiente procedimiento para aislar las neuronas del hipocampo. </li><li> Recoge todas las cortezas disecados en una de 15 ml tubo transparente cónica que contiene 13 ml de frío Hibernate E. Dejar las cortezas cerebrales en el hielo hasta que todas las disecciones se han completado. Debido a su pequeño tamaño, diseccionaron hipocampos se pueden recoger en un tubo Eppendorf de 1,5 ml en lugar de un tubo de 15 ml. Si se desea, en este paso o cortezashipocampos se puede colocar en un vial que contiene criotubo 1 ml de Hibernar E + 2% B27 + gentamicina (50 mg / ml) + Fungizone (250 ng / ml) en la proporción de 2-4 cortezas o 2-4 hipocampos por vial. El tejido cerebral se puede almacenar a 4 ° C en la oscuridad durante hasta una semana (más tarde los tiempos no han sido probados todavía). Cuando sea necesario, utilice unas pinzas finas para transferir el tejido cerebral a un tubo de 15 ml que contiene Hibernate E y luego seguir el protocolo de abajo para aislar a las neuronas. </li><li> Transferir el tubo a una campana de cultivo de tejidos. Permitir las cortezas se depositen en el fondo del tubo y luego retirar con cuidado el sobrenadante. </li><li> Añadir 13 ml de fresca E Hibernar al tubo cónico de 15 ml, permiten cortezas se depositan en el fondo del tubo y retire cuidadosamente el sobrenadante. Repita este paso 2 veces más y, después del último lavado, retire con cuidado todos los medios de comunicación. </li><li> Enzimáticamente digerir cortezas cerebrales mediante la adición de 1-2 ml (dependiendo del número de cortezas; utilizar menos para el aislamiento del hipocampo) de Tr cálidosypLE Express. Sellar la tapa del tubo con Parafilm y flotar el tubo en un baño de agua a 37 ° C durante 10 minutos. </li><li> Rocíe el tubo con etanol al 70% antes de abrir la tapa y añadir 10 ml de Hibernate E. Permita que las cortezas para ubicarse en la parte inferior del tubo y eliminar el sobrenadante. Repita este paso tres veces para vaciar TrypLE Express. En el último paso, retirar con cuidado todos los medios de comunicación. </li><li> Suavemente triturar (4-5 veces) las cortezas en 2 ml de medio completo Neurobasal/B27 utilizando un pulido al fuego Pasteur de vidrio (aproximadamente 1 mm de diámetro). Tenga cuidado para evitar las burbujas. </li><li> Repetir otro 4-5 veces con una estéril Pasteur de vidrio más pequeña pipeta de diámetro (es decir, una pipeta de 1.2 a 3.4 mm de diámetro). No utilice una pipeta Pasteur o menor que este va a destruir las células. </li><li> Permita que las piezas restantes de tejido (por lo general muy pocos, si los hubiera) para resolver. </li><li> Transferir la parte superior de una sola célula suspensión a un nuevo tubo de 15 ml, dejando tras de piezas colocadas de tejido. Pielther diluir la suspensión de células hasta 10-12 ml con Neurobasal/B27 medio completo. </li><li> Mezclar bien y diluir las células para contar mediante la adición de 10 l de suspensión de células a 490 l de solución de conteo 50x (ver tabla III) en un tubo de 1,5 ml de Eppendorf. </li><li> Células de placas sobre placas recubiertas PDL en la densidad de 5,0 x 10 4 / cm 2. Si nucleofection se va a realizar, se recomienda placas las células a una concentración superior (8-10 x 10 4 / cm 2). </li><li> Normalmente, diseccionar 9-10 fetos por experimento, ya que aproximadamente 13 x 10 6 neuronas se derivan de cada feto E17. Si el número de embriones son necesarios, asegúrese de que todo el procedimiento no durará más de dos horas. </li><li> Si se desea, 24 horas después del aislamiento, 10 M de citosina-β-D-arabinofuranosido (AraC) se puede añadir a cada plato, con el fin de prevenir la proliferación glial. Sin embargo, este paso no es necesario ya que Neurobasal/B27 medio inhibe la proliferación glial, Según las recomendaciones del fabricante (Invitrogen / Gibco). </li><li> Las neuronas pueden ser utilizados para los experimentos después de 4-5 días in vitro, aunque el tiempo exacto depende de la etapa diferenciación deseada. Tenemos neuronas cultivadas para hasta 4 semanas sin una disminución significativa en la supervivencia (Figura 1). </li><li> Para el cultivo prolongado, sustituya medio de cultivo cada semana con recién preparada Neurobasal/B27 medio completo. </li></ol> 5. Los resultados representativos Las neuronas cultivadas en cámara de diapositivas de cristal puede ser sometido a inmunocitoquímica. Figura 1 muestra una imagen típica de una neurona cortical fijado después de cinco días de cultivo y immunolabeled con anti-MAP-2 anticuerpo para mostrar los procesos neuronales. La figura 2 muestra una imagen representativa de una neurona del hipocampo de rata después de 3 semanas en cultivo. La morfología neuronal de una célula completamente diferenciada se destaca por el MAP-2 immunolabeling (MAP-2 marcador neuronal, el clon de anticuerpo monoclonal de ratón AP-20, Gene Tex, Irvine, CA), siguiendo un procedimiento estándar que se describió anteriormente 1. Las imágenes se visualizan con la Nikon Eclipse E400 microscopio vertical de fluorescencia equipado con cámara de agua EXI (Qimaging), motor del eje Z, y SlideBook5 adquisición / deconvolución software (Imagen Inteligente Innovations, Inc., Denver, CO). Una serie de imágenes tridimensionales de cada imagen individual se deconvoluted a una imagen bidimensional y se resolvieron mediante el ajuste de la señal de corte a la intensidad máxima cerca de aumentar la resolución. La Figura 3 muestra la pureza de los cultivos neuronales. Proteína lisados ​​se obtuvieron a partir DIV7 cultivos neuronales de rata (CTX) y de un caso de glioblastoma humano (MBG). Como era de esperar, el lisado neuronal es muy positivo para la proteína neuronal MAP-2 y negativa para el marcador de astrocitos GFAP, mientras que el lisado de proteínas GBM es negativa fo MAP-2 y positivas para GFAP. Aunque en el protocolo que hemos estado usando Hibernate E durante varios años como medio de disección y aclarado, recientemente, hemos explorado un uso adicional y muy práctica de la misma para preservar los tejidos del cerebro para su uso posterior. La Figura 4 muestra un par de días in vitro 5 (DIV5) la cultura de las neuronas corticales de rata aisladas de cortezas mantenerse a 4 ° C durante una semana en Hibernate E + B27 después de la disección original de los embriones. Las neuronas se cultivaron sobre un vidrio de dos cámaras de diapositivas recubierta con PDL y laminina como se describió previamente. La imagen adquirida se deconvoluted utilizando SlideBook5 adquisición / software de deconvolución como se ha descrito anteriormente (Figura 2). <img alt="Figura 1" src="/files/ftp_upload/3965/3965fig1.jpg" /> Figura 1. Imagen representativa de una neurona cortical nucleofected con pmaxGFP (Amaxa, Lonza, Walkersville, MD) y immunolabeled con MAPA-2 De anticuerpos, de color rojo. Magnificación original 100x. <img alt="Figura 2" src="/files/ftp_upload/3965/3965fig2.jpg" /> Figura 2. Imagen representativa muestra MAP-2 inmunomarcación, en rojo, de las neuronas del hipocampo después de 3 semanas en cultivo. DAPI, en azul, muestra los núcleos celulares. Magnificación 40x original. <img alt="Figura 3" src="/files/ftp_upload/3965/3965fig3.jpg" /> Figura 3. Western blot que muestra la pureza de los cultivos celulares neuronales. 30 g de proteína lisados ​​de rata y humanos neuronales MBG se separaron por electroforesis y se sometió a análisis Western blot siguiendo procedimientos estándar 1. Anti-MAP-2 era un conejo policlonal de Señalización Celular (Danvers, MA), anticuerpo anti-GFAP era un anticuerpo monoclonal de ratón de Chemicon (Millipore, Billerica, MA), y el anticuerpo monoclonal de ratón anti-GRB2 anticuerpo era de Transduction Laboratories (BD Sparks, MD). GRB2 se usó como un control de carga. </p&gt; <img alt="Figura 4" src="/files/ftp_upload/3965/3965fig4.jpg" /> Figura 4. Imágenes representativas de los cinco días in vitro (DIV5), las neuronas corticales de rata obtenidos a partir de cortezas que quedan en hibernación E + B27 a 4 ° C durante una semana después de la disección. A) Fase de contraste de las neuronas cultivadas en un vaso de doble cámara de diapositivas. Magnificación original 20X. B) La inmunofluorescencia muestra la expresión de MAP-2 en los procesos neuronales, en verde, el cultivo fue negativo para el marcador de astrocitos GFAP. DAPI, en azul, indica que los núcleos celulares. Magnificación 40x original.

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El método de disección y cultivo de las neuronas del hipocampo de rata y cortical aquí descrito permite la realización de experimentos utilizando cultivos neuronales casi puros cultivadas en un medio químicamente definido (Figura 3). Aunque los protocolos para el cultivo de las neuronas casi puros en medios libres de suero han sido descritas previamente 2,3,4, hay cambios importantes realizados en nuestro método. A diferencia de los protocolos tradicionales (es decir, Banker et al.) <su...

<table border="1"><tbody><tr><td> Reactivo </td><td> Concentración </td></tr><tr><td> Neurobasal </td><td> 98% </td></tr><tr><td> B27 </td><td> 2% </td></tr><tr><td> Glutamax </td><td> 0,5 mM </td></tr></tbody></table> Tabla I. Neurobasal/B27 medio completo. <table border="1"><tbody><tr><td> Reactivo </td><td> Concentración </td></tr><tr><td> Glucosa </td><td> 16 mM </td></tr><tr><td> Sacarosa </td><td> 22 mM </td></tr><tr><td> HEPES </td><td> 10 mM </td></tr><tr><td> NaCl </td><td> 160 mM </td></tr><tr><td> KCl </td><td> 5 mM </td></tr><tr><td> Na 2 HPO 4 </td><td> 1 mM </td></tr><tr><td> KH 2 PO 2 </td><td> 0,22 mM </td></tr><tr><td> Gentamicina </td><td> 50 g / ml </td></tr&gt; <tr><td> Fungizone </td><td> 250 ng / ml </td></tr><tr><td> pH </td><td> 7.4 </td></tr><tr><td> Osmolaridad </td><td> 320-330 mOsm </td></tr></tbody></table> Tabla II. Disección medio. <table border="1"><tbody><tr><td> Reactivo </td><td> Volumen (l) </td></tr><tr><td> Neurobasal/B27 medio completo </td><td> 240 </td></tr><tr><td> Trypan mancha azul del 0,4% </td><td> 250 </td></tr><tr><td> Total </td><td> 490 </td></tr></tbody></table> Tabla III. Contando 50x solución. <table border="1"><tbody><tr><td> Reactivo </td><td> Empresa </td><td> Cat. número </td></tr><tr><td> Hibernate E </td><td> Brainbits </td><td> 767171 </td></tr><tr><td> Neurobasal </td><td> Gibco, Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 </td><td> Gibco, Invitrogen </td><td> 17504-044 </td></tr><tr><td> Fungizone </td><td> Gibco, Invitrogen </td><td> 15290-018 </td></tr><tr><td> La gentamicina sulfato </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> Glutamax 200 mM </td><td> Gibco, Invitrogen </td><td> 35050 </td></tr><tr><td> TrypLE expreso w / o rojo fenol </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Citosina-β-D-arabinofuranosido clorhidrato de </td><td> Sigma Aldrich </td><td> C6645 </td></tr><tr><td> Poli-D-lisina </td><td> Sigma Aldrich </td><td> P6407 </td></tr><tr><td> La laminina 1 mg / ml </td><td> Millipore </td><td> CC095 </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3375 </td></tr><tr><td> Trypan mancha azul del 0,4% </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tabla IV. Reactivos específicos. <table border="1"><tbody><tr><td> Equipo </td><td> Empresa </td><td> Cat. número </td></tr><tr><td> Microscopio estéreo </td><td> Olimpo </td><td> SZ61 </td></tr><tr><td> Pinzas Grandes </td><td> FST </td><td> 11022-14 </td></tr><tr><td> Una pinza fina </td><td> Moria </td><td> MC40B </td></tr><tr><td> Micro pinzas de punta fina </td><td> Moria </td><td> MC31 </td></tr><tr><td> Afiladas tijeras </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Micro tijeras de disección </td><td> FST </td><td> 91460-11 </td></tr><tr><td> Micro Tijeras de disección curvas </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Vidrio 2-cámara portaobjetos </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> Placas de 60 mm </td&gt; <td> BD Falcon </td><td> 353002 </td></tr><tr><td> Placas de 100 mm </td><td> Corning </td><td> 430167 </td></tr><tr><td> 15 ml tubos </td><td> BD Falcon </td><td> 352099 </td></tr><tr><td> 1,5 ml de la crio-tubo vial </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Tabla V. Equipos específicos.

isolation and culture of rat embryonic neural cells: a quick protocol

Estamos describiendo un método rápido para disociar y la cultura de las neuronas del hipocampo o la corteza a partir de embriones de rata E15-17. El procedimiento se puede aplicar con éxito para el aislamiento de ratón y humanos neuronas primarias y progenitores neurales. Neuronas disociadas se mantienen en medio libre de suero hasta varias semanas. Estos cultivos pueden ser utilizados para nucleofection, inmunocitoquímica, la preparación de ácidos nucleicos, así como la electrofisiología. Mayores cultivos neuronales también pueden ser transfectadas con una tasa de eficiencia bueno por transducción lentiviral y, menos eficiente, con fosfato de calcio o basados ​​en lípidos métodos tales como lipofectamina.

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Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Se describe un método rápido para aislar y cultivar las neuronas del hipocampo y la corteza a partir de embriones de roedores. Este protocolo nos permite realizar experimentos en los que los cultivos neuronales casi puros son necesarios.

Aislamiento y cultivo de células neuronales de ratas embrionarias: un protocolo rápido

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

isolation-and-culture-of-rat-embryonic-neural-cells-a-quick-protocol

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38.0K vistas.  LSU Health Sciences Center - New Orleans. Se describe un método rápido para aislar y cultivar las neuronas del hipocampo y la corteza a partir de embriones de roedores. Este protocolo nos permite realizar experimentos en los que los cultivos neuronales casi puros son necesarios.

Video: Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Investigación

Neurociencias

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

El aislamiento y la expansión de las células troncales nerviosas de ratones adultos mediante el ensayo de neuroesfera

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

El establecimiento de embriones de ratón con células madre neurales Cultura mediante el ensayo de neuroesfera

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Aislamiento y cultivo de células troncales de la cresta neural de los folículos del cabello humano

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Lectina basado en Aislamiento y cultivo de motoneuronas embrionarias de ratón

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Aislamiento y cultivo de células humanas las papilas fungiformes gusto

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Aislamiento y cultivo de células de la cresta neural de embriones murinos del Tubo Neural

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Disección, Cultura y Análisis de<em&gt; Xenopus laevis</em&gt; Tejido de la retina embrionaria

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Aislamiento y cultivo de células endoteliales del cerebro anterior embrionarias

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Un ratón, dos culturas: Aislamiento y cultivo de adultos Neural las células madre de las dos zonas neurogénicos de ratones individuales

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...