Con el apoyo de la Fundación de Becas de Postgrado Fazit a SS, el Ministerio Federal de Educación e Investigación (BMBF 01 EO 0803) en KB y la Comisión Europea FP7 subvención PIRG08-GA-2010 a 276.989, Neurex, y la beca de la Fundación Alemana para la Investigación SCHA 1442 / 3-1 ante el CS Los autores no tienen intereses financieros en conflicto.

1. El aislamiento y forro de una mezcla de células corticales Aislamiento de células mixtas para cultivos de astrocitos corticales se puede realizar utilizando P1 a P4 crías de ratón. Con el fin de conseguir una densidad de astrocito adecuado, es necesario el uso de 4 capas corticales de ratón pup por T75 matraz de cultivo de tejido. Por lo tanto, los volúmenes en el protocolo siguiente se calculan para una preparación de células usando 4 crías de ratón. <ol><li> Antes de iniciar el procedimiento de disección, precalentamiento 30 ml de medios de cultivo de astrocitos (DMEM, alta glucosa + 10% inactivado por calor suero fetal bovino + 1% de penicilina / estreptomicina; véase la tabla) a 37 ° C. Cubra un matraz T75 con 20 ml de poli-D-lisina (PDL) a una concentración de 50 mg / ml en agua de grado de cultivo de células durante 1 hora a 37 ° C en la incubadora de CO 2. </li><li> Para el procedimiento de disección, preparar todos los reactivos y materiales necesarios. Usted necesitará: tijeras quirúrgicas, pinzas finas lisas, pinzas de punta plana, toallas de papel, bolsas de residuos, un 70% de etanold 2 platos de disección (3,5 cm de diámetro) en el hielo llena con 2 ml de HBSS cada uno. </li><li> Aplique suavemente y rociar la cabeza y el cuello del ratón cachorro con 70% de etanol. El animal es sacrificado por decapitación con las tijeras. </li><li> Realizar una incisión en línea media, de posterior a anterior, a lo largo del cuero cabelludo para revelar el cráneo. </li><li> Cortar cuidadosamente el cráneo desde el cuello hasta la nariz. Dos cortes adicionales se realizan para permitir el acceso adicional al cerebro: El primer corte se hace anterior de los bulbos olfatorios, la otra inferior del cerebelo para desconectar el cráneo de la base del cráneo. </li><li> Uso de las pinzas de punta plana, las aletas craneales se volcó suavemente a un lado y el cerebro se extrae y se coloca en el plato primero disección llena con HBSS. Coloque el plato de nuevo en hielo y seguir cosechando los 4 cerebros. </li><li> El resto del procedimiento de disección se realiza bajo un estereomicroscopio. En primer lugar, los bulbos olfatorios y el cerebelo se eliminan mediante la multafórceps de disección (Figura 1B). </li><li> Con el fin de recuperar las cortezas, agarra el extremo posterior del cerebro con las pinzas finas, realizar una incisión en línea media entre los hemisferios, insertar un segundo conjunto de pinzas de la arboleda creado y se desprenden de la estructura en forma de placa de la corteza de la cerebro. </li><li> Diseccionar cuidadosamente las meninges de la corteza de los hemisferios tirando con las pinzas finas (Figura 1 D &#39;). Este paso evita la contaminación del cultivo de astrocitos final por células de las meninges y los fibroblastos. Transfiera los hemisferios corticales preparados en el segundo plato lleno de HBSS y devolverlo en hielo. Continuar en consecuencia con todas las 4 capas corticales. </li><li> Por último, cortar cada hemisferio corteza en trozos pequeños con cuchillas afiladas (aproximadamente 4 a 8 veces). </li><li> En condiciones estériles, piezas de transferencia de corteza en un tubo Falcon de 50 ml y añadir HBSS hasta un volumen total de 22,5 ml. </li><li> Añadir 2,5 ml de tripsina al 2,5%, mezclar e incubarel tejido en el baño de agua a 37 ° C durante 30 min. Mezclar por agitación ocasional cada 10 min. </li><li> Centrifugar durante 5 min a 300 xg para piezas de pellets tejido de la corteza. </li><li> Retire con cuidado el sobrenadante por decantación. Con el fin de evitar la pérdida del sedimento de tejido que puede retener mecánicamente usando una pipeta. Disociar el tejido en una suspensión de células individuales mediante la adición de 10 ml de medio de recubrimiento de astrocitos y pipeteado vigoroso usando una pipeta de plástico de 10 ml hasta trozos de tejido se disocia en células individuales (20 a 30 veces). Ajuste el volumen a 20 ml con medio chapado astrocitos. Puede prueba de la disociación del tejido de la corteza en células individuales mediante recuento usando un hematocitómetro. Una preparación de 4 cortezas de ratón cachorro debe ceder 10-15 x 10 6 células individuales disociados. </li><li> Aspirar PDL del matraz de cultivo T75, placa de la suspensión disociado sola célula y se incuba a 37 ° C en la incubadora de CO 2. </li></ol> 2. Obtención de una EnrCultura Astrocyte iched <ol><li> Cambiar el medio 2 días después de la siembra de las células mixtas corticales y los 3 días posteriormente. </li><li> Después de 7 a 8 días, cuando los astrocitos son confluentes microglia y superposición sentarse expuestos en la capa de astrocitos o que ya están separados de la capa de astrocitos (Figura 2), agitar el matraz T75 a 180 rpm durante 30 min en un agitador orbital para eliminar la microglía. Desechar el sobrenadante que contiene microglia o si desea examinar la cultura y la microglia, lo hace girar hacia abajo y la placa de la cultura 28,29. </li><li> Añadir 20 ml de medio fresco cultivo de astrocitos y continuar agitando el matraz a 240 rpm durante 6 horas para eliminar las células precursoras de oligodendrocitos (OPC). Dado que algunos OPC no se desprenderá completamente de la capa de astrocitos, siguen agitar fuertemente durante 1 min con el fin de evitar la contaminación OPC. Desechar el sobrenadante o girar hacia abajo y la placa, si lo desea con la cultura OPC 29. </li><li> Enjuague el conflue restantent capa de astrocitos dos veces con PBS, aspirar el PBS, añadir 5 ml de tripsina-EDTA y se incuban en la incubadora de CO 2 a 37 ° C. Compruebe la separación de los astrocitos cada 5 minutos y hacer cumplir la separación de los astrocitos golpeando el frasco en la palma de tu mano (2-3 veces). </li><li> Después de astrocitos se desprenden del matraz de cultivo, añadir 5 ml de medio de cultivo de astrocitos, células giran a 180 xg durante 5 min, aspirar el sobrenadante y añadir 40 ml de medio fresco chapado de astrocitos. Uno T75 frasco de cultivo de tejidos deben producir alrededor de 1x10 6 células después de la división celular en primer lugar. Células de la placa en dos matraces T75 de cultivo y se incuba a 37 ° C en la incubadora de CO 2. Cambiar el medio cada 2 a 3 días. </li><li> 12-14 días después de la primera división de los astrocitos se sembraron en la concentración apropiada de células 24-48 horas antes de realizar el experimento. Uno T75 frasco de cultivo de tejidos deben producir alrededor de 1.5-2 x 10 6 células después de la división celular segundo. </li></ol>

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El método descrito aquí se basa en la preparación de cultivo de astrocitos a partir de cerebros de roedores neonatos, originalmente descrito por McCarthy y de Vellis en 1980 27. El método modificado del aislamiento y el cultivo de astrocitos corticales a partir de P1 a P4 postnatal cerebro de ratón se presenta aquí es rápido, los rendimientos de los astrocitos primarios puros y es altamente reproducible. Esta técnica se puede transferir fácilmente para aislar los astrocitos a partir de otras especies...

Los astrocitos son un tipo de célula muy abundantes en el sistema nervioso central (SNC). La relación de los astrocitos de las neuronas es de 1:3 en la corteza de ratones y ratas, mientras que hay 1,4 astrocitos por neurona en la corteza humana 1. El interés en función de los astrocitos se ha incrementado dramáticamente en los últimos años. Una función clave de los astrocitos es su papel en la prestación de apoyo estructural y metabólico de las neuronas 2,3. Papeles recién descubiertas de astrocitos cubren un amplio espectro de funciones. Estos incluyen guiar la migración de los axones en desarrollo y los neuroblastos determinadas durante el desarrollo de 4-6, funciones en la transmisión sináptica, la fuerza de sinapsis y procesamiento de la información por los circuitos neuronales 7-9, roles en la barrera hematoencefálica (BBB) ​​formación 10 y la integridad 11-13 y la regulación del tono vascular cerebral 14. Otra característica importante de los astrocitos es su respuesta a la lesión. Bajo condiciones patológicas astrocytES vuelven reactivos y más regular al alza la expresión de la proteína de filamentos intermedios ácida fibrilar glial (GFAP) y proteínas inhibidoras de la matriz extracelular (ECM) 15,16. Astrocitos reactivos demarcar el sitio de la lesión del tejido sano mediante la formación de una cicatriz glial, que consiste principalmente en astrocitos proteínas secretadas de ECM de la proteoglicano sulfato de condroitina (CSPG) de la familia, los principales factores que inhiben la regeneración axonal después de una lesión del SNC 15-17. Los astrocitos se originan en gliales radiales (RG) de las células durante la embriogénesis tardía y la vida postnatal temprana. Después de especificación de astrocitos se ha producido, precursores de astrocitos migran a sus posiciones finales, en el que comienzan el proceso de diferenciación terminal. In vivo, los astrocitos parecen ser maduro tres a cuatro semanas después del nacimiento, como se indica por su morfología típica de 18,19. Una subpoblación de células RG convertir en astrocitos de la zona subventricular (tipo de células B). Both, RG y células de tipo B funcionar como astrocitos como células madre neurales (NSC) durante el desarrollo y en el adulto, respectivamente. Al igual que los astrocitos, RG y células de tipo B también expresan el transportador de glutamato específico de astrocitos (GLAST), cerebro lípido-proteína de unión (BLBP), y GFAP, lo que indica que estos marcadores no pueden ser exclusivamente utilizado para etiquetar específicamente astrocitos adultos. En contraste con los astrocitos adultos parenquimatosas, que no se dividen en el cerebro sano RG, y el tipo B muestra células tallo potencial de la célula tales como la capacidad de auto-renovación. La desregulación de los astrocitos se ha implicado en numerosas patologías, incluyendo la enfermedad de Alzheimer 20,21, enfermedad de Huntington 22, 23 enfermedad de Parkinson, síndrome de Rett 24 y enfermedad de Alexander 25. Por otra parte, los astrocitos reaccionar a todos los insultos del SNC, que conduce a la activación de astrocitos y la formación de cicatriz glial astrocytic 16,26. La cicatriz glial astrocytic que forma tr cerebro siguienteauma lesión o la médula espinal se piensa que es la barrera primer impedir la regeneración neuronal 15. El desarrollo de métodos fiables para aislar y mantener las poblaciones purificadas de células ha sido esencial para nuestra comprensión del sistema nervioso. Los trabajos pioneros de McCarthy y de Vellis permite a los investigadores hasta la fecha para preparar cultivos casi puros de astrocitos de rata neonatal tejido 27. Se ha aprendido mucho sobre la biología de los astrocitos utilizando este método, que se presenta aquí en una forma ligeramente modificada para aislar los astrocitos corticales de ratón. Como complemento de los estudios in vivo, los astrocitos, así como el medio condicionado obtenido utilizando el descrito en el cultivo in vitro, son herramientas valiosas para obtener más ideas sobre las funciones de los astrocitos.

<table border="1"><tbody><tr><td> Nombre de la solución de trabajo </td><td> Empresa </td><td> Número de catálogo </td><td> Concentración final </td></tr><tr><td></td><td></td><td></td><td> Medios de cultivo de astrocitos </td></tr><tr><td> DMEM, alta glucosa </td><td> Life Technologies </td><td> 31966-021 </td><td></td></tr><tr><td> FBS, inactivado por calor </td><td> Life Technologies </td><td> 10082-147 </td><td> Concentración final: 10% </td></tr><tr><td> Penicilina-Estreptomicina </td><td> Life Technologies </td><td> 15140-122 </td><td> Concentración final: 1% </td></tr><tr><td></td><td></td><td></td><td> Solución para la excavación del tejido cerebralestion </td></tr><tr><td> HBSS </td><td> Life Technologies </td><td> 14170-088 </td><td></td></tr><tr><td> 2,5% de tripsina </td><td> Life Technologies </td><td> 15090-046 </td><td> Concentración final: 0,25% </td></tr><tr><td></td><td></td><td></td><td> Otro </td></tr><tr><td> 70% (vol / vol) de etanol </td><td> Roth </td><td> 9065.2 </td><td></td></tr><tr><td> Poli-D-lisina </td><td> Millipore </td><td> A-003-E </td><td> 50 ug / ml </td></tr><tr><td> Agua </td><td> PAA </td><td> S15-012 </td><td> grado celda de la cultura </td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td> grado celda de la cultura </td></tr><tr><td> Trate de 0,05%psin-EDTA </td><td> Life Technologies </td><td> 25300-062 </td><td></td></tr><tr><td> 0,45 filtro estéril micras </td><td> Sartorio </td><td> 16555 </td><td></td></tr><tr><td> 3,5 cm plato de Petri </td><td> BD Falcon </td><td> 353001 </td><td></td></tr><tr><td> 15 ml Falcon tubo </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> 50 ml Falcon tubo </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> 75 cm 2 matraz de cultivo tisular </td><td> BD Falcon </td><td> 353136 </td><td></td></tr><tr><td> Pinzas, bien </td><td> Dumont </td><td> 2-1032, 2-1033 </td><td> # 3c, # 5 </td></tr><tr><td> Pinzas, punta plana </td><td> KLS Martin </td><td> 12-120-11 </td><td></td></tr><tr><td> 13 cm tijeras quirúrgicas </td><td> Aesculap </td><td> BC-140-R </td><td></td></tr><tr><td> Estereomicroscopio </td><td> Leica </td><td></td><td> MZ7.5 </td></tr><tr><td> Estereomicroscopio + cámara </td><td> Leica </td><td></td><td> MZ16F; DFC320 </td></tr><tr><td> Microscopio con cámara + </td><td> Zeiss, Canon </td><td></td><td> Primo Vert; PowerShot A650 IS </td></tr><tr><td> Centrífugo </td><td> Eppendorf </td><td> 5805000.017 </td><td> Centrifuge5804R </td></tr><tr><td> Orbital Shaker </td><td> Thermo Scientific </td><td> SHKE 4450-1CE </td><td> MaxQ 4450 </td></tr><tr><td> Baño de agua </td><td> Julabo </td><td></td><td> SW20, 37 ° C </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

Los astrocitos son un tipo de células abundante en el cerebro de los mamíferos, pero aún queda mucho por aprender acerca de sus características moleculares y funcionales. Astrocitos in vitro de sistemas de cultivo celular se puede utilizar para estudiar las funciones biológicas de estas células gliales en detalle. Este protocolo video muestra cómo obtener los astrocitos puras mediante el aislamiento y la cultura de una mezcla de células corticales de crías de ratón. El método se basa en la ausencia de neuronas viables y la separación de los astrocitos, los oligodendrocitos y microglia, las tres poblaciones principales de células gliales del sistema nervioso central, en cultivo. Imágenes representativas durante los primeros días de cultivo demostrar la presencia de una población de células mixtas e indicar el punto de tiempo, cuando los astrocitos se hacen confluentes y debe ser separado de microglia y oligodendrocitos. Por otra parte, se demuestra la pureza y la morfología astrocytic de astrocitos en cultivo utilizando tinciones inmunocitoquímicas para bien establecidos yrecién descrito marcadores de astrocitos. Este sistema de cultivo puede ser fácilmente utilizado para obtener los astrocitos puros ratón y el medio acondicionado de astrocitos para el estudio de diversos aspectos de la biología de astrocitos.

Tras el aislamiento del cerebro de ratón completo (Figura 1A), el cerebelo y el bulbo olfativo tiene que ser eliminado (Figura 1B). Las cortezas se pelan del vástago de cerebro de ratón (Figura 1C) y de las meninges de la corteza individual (Figura 1D &#39;) se retiran cuidadosamente (Figura 1E). Meninges son evidentes por el sistema de la arteria meníngea y resultados incompletos de eliminación de contaminación en el cultivo de a...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

Isolation and Culture of Mouse Cortical Astrocytes

Los astrocitos se han reconocido para ser versátiles células que participan en los procesos biológicos fundamentales que son esenciales para el desarrollo normal del cerebro y la función, y la reparación del sistema nervioso central. Aquí presentamos un procedimiento rápido para obtener cultivos puros de astrocitos de ratón para estudiar la biología de esta clase importante de centrales células del sistema nervioso.

Aislamiento y cultivo de astrocitos corticales de ratones

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

isolation-and-culture-of-mouse-cortical-astrocytes

Research

JoVE Journal

Neuroscience

90.8K vistas.  University of Freiburg. Los astrocitos se han reconocido para ser versátiles células que participan en los procesos biológicos fundamentales que son esenciales para el desarrollo normal del cerebro y la función, y la reparación del sistema nervioso central. Aquí presentamos un procedimiento rápido para obtener cultivos puros de astrocitos de ratón para estudiar la biología de esta clase importante de centrales células del sistema nervioso.

Video: Isolation and Culture of Mouse Cortical Astrocytes

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

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. ...

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 ...

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 ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelado astrocitoma Patogénesis<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Uso cortical astrocitos o células troncales nerviosas de ratones condicionales, Genéticamente Manipulados

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Diferencias de sexo en el ratón del hipocampo después de astrocitos<em&gt; In-Vitro</em&gt; isquemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

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 ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Diferenciación de células madre embrionarias de ratón en precursores corticales interneurona

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.