Apoiado pela Fundação Fazit graduação bolsa para SS, o Ministério Federal da Educação e Pesquisa (BMBF 01 OE 0803) para KB e da Comissão Europeia FP7 Grant PIRG08-GA-2010-276989, Neurex, ea Fundação Alemã de Pesquisa Grant SCHA 1442 / 3-1 para CS Os autores não têm conflitos de interesses financeiros.

1. Isolamento e Chapeamento de Mixed células corticais Isolamento celular mista cortical para culturas de astrócitos podem ser realizados utilizando P1 a P4 crias de rato. Para alcançar densidade adequada dos astrócitos é necessário utilizar quatro córtices pup rato por frasco de cultura de tecidos T75. Por conseguinte, os volumes no protocolo que se segue são calculados para uma preparação de células com 4 crias de rato. <ol><li> Antes de iniciar o procedimento de dissecção, 30 ml de Pré-aquecer os meios de cultura de astrócitos (DMEM, glucose elevada + 10% de soro fetal de soro de bovino a 1% + Penicilina / Estreptomicina, ver tabela) a 37 ° C. Um casaco frasco T75 com 20 ml de poli-D-lisina (PDL), a uma concentração de 50 ug / ml em água de grau de cultura de células durante 1 hora a 37 ° C na incubadora de CO2. </li><li> Para o procedimento de dissecção, preparar todos os reagentes e materiais necessários. Você vai precisar de: uma tesoura cirúrgica, lisas pinça fina, pinças ponta plana, toalhas de papel, saco de lixo, 70% de etanol umd 2 pratos de dissecação (3,5 cm de diâmetro) sobre gelo enchido com 2 ml de HBSS a cada. </li><li> Segurar delicadamente e pulverizar a cabeça e pescoço do rato filhote com etanol 70%. O animal é sacrificado por decapitação utilizando uma tesoura. </li><li> Realizar uma incisão na linha média, posterior para anterior, ao longo do couro cabeludo para revelar o crânio. </li><li> Corte o crânio cuidadosamente desde o pescoço até o nariz. Dois cortes adicionais são realizados para permitir o acesso ainda mais para o cérebro: O primeiro corte é feito anterior dos bulbos olfatórios, o outro inferior do cerebelo para desconectar o crânio da base do crânio. </li><li> Usando a pinça de ponta plana, as abas cranianas são suavemente virado para o lado e o cérebro é retirado e colocado no primeiro prato de dissecação cheio com HBSS. Coloque o prato de volta no gelo e continuar a colher todos os quatro cérebros. </li><li> O resto do procedimento de dissecção é realizada sob um estereomicroscópio. Em primeiro lugar, os bulbos olfativos e no cerebelo são removidos usando o finofórceps de dissecção (Figura 1B). </li><li> A fim de recuperar o córtex, pegue a extremidade posterior do cérebro com a pinça fina, realizar uma incisão entre os hemisférios, inserir um segundo conjunto de pinça para o bosque criado e retire a estrutura da placa-como o córtex do cérebro. </li><li> Dissecar cuidadosamente as meninges dos hemisférios corticais, puxando com o fórceps finos (Figura 1D). Este passo evita a contaminação da cultura final do astrócito por células meníngeas e fibroblastos. Transfira os hemisférios corticais preparadas para o segundo prato cheio com HBSS e devolvê-lo em gelo. Continuar de acordo com todos os 4 cortices. </li><li> Finalmente, cortar cada hemisfério do córtex em pequenos pedaços, utilizando lâminas cortantes (cerca de 4 a 8 vezes). </li><li> Sob condições estéreis, pedaços de córtex de transferência para um tubo Falcon de 50 ml e adicionar HBSS para um volume total de 22,5 ml. </li><li> Adicionar 2,5 ml de 2,5% de tripsina, misturar e incubaro tecido no banho de água a 37 ° C durante 30 min. Misturar por agitação ocasional cada 10 min. </li><li> Centrifugar durante 5 minutos a 300 xg para peças de tecido do córtex da pelota. </li><li> Remova cuidadosamente o sobrenadante por decantação. A fim de evitar a perda do tecido pellet pode ser mecanicamente retê-lo usando uma pipeta. Dissociar o tecido numa única suspensão de células por adição de 10 ml de meio de revestimento de astrócitos e pipetagem vigorosa com uma pipeta de plástico de 10 ml até pedaços de tecido são dissociadas em células individuais (20 a 30 vezes). Ajustar o volume a 20 ml com o meio de revestimento de astrócito. Pode prova a dissociação do tecido do córtex em células individuais por contagem usando um hematocytometer. Uma preparação de 4 córtices rato filhote deve render 10-15 x10 6 dissociadas células individuais. </li><li> Aspirar PDL a partir do balão de cultura T75, a placa de suspensão de células dissociadas único e incubar a 37 ° C na incubadora de CO2. </li></ol> 2. Obtenção de um EnrCultura astrocitária iched <ol><li> Altere as médias 2 dias após o plaqueamento das células corticais e mistas todos os 3 dias seguintes. </li><li> Depois de 7-8 dias, quando os astrócitos microglia são confluentes e sobreposição sentar exposta sobre a camada de astrócitos são ou já separado da camada de astrócitos (Figura 2), agitar o frasco T75 a 180 rpm durante 30 min num agitador orbital a remover microglia. Descartar o sobrenadante contendo microglia ou se deseja para a cultura e examinar microglia, girá-lo para baixo e placa para cultura de 28,29. </li><li> Adicionam-se 20 ml de meio de cultura fresco astrócitos e continuar agitando o frasco a 240 rpm durante 6 h para remover as células precursoras de oligodendrócitos (OPC). Uma vez que alguns OPCs não destacar completamente a partir da camada de astrócitos, continuar a agitar vigorosamente à mão durante 1 minuto, a fim de evitar a contaminação do OPC. Descartar o sobrenadante ou girá-lo para baixo e placa, se quiser cultura OPCs 29. </li><li> Lave o restante confluent camada de astrócitos duas vezes com PBS, aspirar o PBS, adicionar 5 ml de tripsina-EDTA e incubado no incubador de CO2 a 37 ° C. Verifique destacamento de astrócitos a cada 5 minutos e fazer cumprir destacamento de astrócitos por bater o frasco contra a palma da sua mão (2-3 vezes). </li><li> Depois de astrócitos são separadas do frasco de cultura, adicionam-se 5 ml de meio de cultura de astrócitos, células de rotação a 180 xg durante 5 min, aspirar o sobrenadante e adicionar 40 ml de meio de revestimento fresco de astrócitos. Um frasco de cultura de tecidos T75 deve render cerca de 1x10 6 células após o desdobramento primeira célula. As células da placa em dois frascos de cultura T75 e incubar a 37 ° C na incubadora de CO2. Alterar o meio a cada 2 a 3 dias. </li><li> 12-14 dias após a primeira divisão astrócitos são plaqueadas na célula concentração adequada 24-48 horas antes de realizar a experiência. Um frasco de cultura de tecidos T75 deve render cerca de 1,5-2 x10 6 células após a separação segunda célula. </li></ol>

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O método descrito aqui é baseado na preparação da cultura de astrócitos de cérebros de roedores neonatais, originalmente descritos por McCarthy e Vellis de, em 1980, 27. O método de modificação do isolamento e da cultura de astrócitos corticais a partir de P1 a P4 pós-natal do cérebro de rato aqui apresentado é rápido, rendimentos puros astrócitos primários e é altamente reprodutível. Esta técnica pode ser facilmente transferido para isolar os astrócitos de outras espécies, tais como o ra...

Os astrócitos são um tipo de célula muito abundantes no sistema nervoso central (SNC). A razão entre os astrócitos de neurónios é 1:3 no córtex de ratos e ratazanas, enquanto que existem 1,4 astrócitos por neurónios no córtex humano 1. Interesse em função de astrócitos aumentou dramaticamente nos últimos anos. A função principal de astrócitos é o seu papel no apoio estrutural e metabólica dos neurônios 2,3. Papéis recém-descobertos para astrócitos abrangem um amplo espectro de funções. Estes incluem guiar a migração de axónios em desenvolvimento e neuroblastos determinados durante o desenvolvimento 4-6, a formação de funções na transmissão sináptica, sinapse força e de processamento de informações por circuitos neurais 7-9, papéis na barreira hemato-encefálica (BHE) 10 e 11-13 integridade e regulação do tônus ​​vascular cerebral 14. Outra característica importante dos astrócitos é a sua resposta a lesões. Sob astrocyt condições patológicases tornar reactivo e mais regular positivamente a expressão do filamento intermediário glial da proteína fibrilar ácida (GFAP) e inibidores da matriz extracelular (ECM) proteínas 15,16. Astrócitos reativos demarcar o local da lesão do tecido saudável através da formação de uma cicatriz glial, que consiste principalmente de astrócitos proteínas secretadas ECM do sulfato de condroitina proteoglicanos família (CSPG), os principais fatores que inibem a regeneração axonal após lesão do SNC 15-17. Os astrócitos são originários de radiais gliais (RG), as células durante a embriogênese tarde e vida pós-natal precoce. Após a especificação dos astrócitos ocorreu, precursores de astrócitos migram para as suas posições finais, em que começa o processo de diferenciação terminal. In vivo, os astrócitos parecem estar maduro três a quatro semanas após o nascimento, tal como indicado pela sua morfologia típica 18,19. Uma subpopulação de células de RG converter em astrócitos zona subventricular (do tipo células B). Both RG, e as células do tipo B funcionam como astrócitos do tipo de células estaminais neurais (NSC) durante o desenvolvimento e no adulto, respectivamente. Como astrócitos RG, e as células do tipo B também expressam o transportador glutamato astrócito-específico (GLAST), proteína de ligação ao lípido cérebro (BLBP) e GFAP, indicando que estes marcadores não pode ser exclusivamente utilizado para rotular os astrócitos especificamente adultos. Em contraste com os adultos astrócitos parenquimatosas, que não se dividem no cérebro saudável, RG e B do tipo células exibem potencial das células estaminais, tais como a capacidade de se auto-renovar. A desregulação dos astrócitos tem sido implicada em numerosas patologias, incluindo a doença de Alzheimer 20,21, doença de Huntington, 22, doença de Parkinson 23, 24 e síndrome de Rett doença de Alexander 25. Além disso, os astrócitos reagir a todos os insultos do SNC, levando à activação de astrócitos e a formação de cicatriz glial astrocítica 16,26. A cicatriz glial astrocitária que forma tr cérebro seguinteauma lesão da medula espinhal ou é pensado para ser a principal barreira impedindo a regeneração neuronal 15. O desenvolvimento de métodos fiáveis ​​para isolar e manter populações puras de células tem sido essencial para a compreensão do sistema nervoso. Trabalho pioneiro de McCarthy e de Vellis permite investigadores a data para preparar culturas quase puras de astrócitos do tecido de ratos neonatos 27. Muito tem sido aprendido sobre a biologia astrócitos usando este método, que é apresentado aqui em uma forma ligeiramente modificada para isolar astrócitos de rato corticais. Complementando os estudos in vivo, astrócitos, bem como meio condicionado obtido usando o descrito na cultura in vitro, são ferramentas valiosas para continuar a obter insights sobre funções de astrócitos.

<table border="1"><tbody><tr><td> Nome da solução de trabalho </td><td> Companhia </td><td> Número de catálogo </td><td> Concentração final </td></tr><tr><td></td><td></td><td></td><td> Astrócitos meios de cultura </td></tr><tr><td> DMEM, glucose elevada </td><td> Life Technologies </td><td> 31966-021 </td><td></td></tr><tr><td> FBS, inactivado pelo calor </td><td> Life Technologies </td><td> 10082-147 </td><td> A concentração final: 10% </td></tr><tr><td> Penicilina-Estreptomicina </td><td> Life Technologies </td><td> 15140-122 </td><td> A concentração final: 1% </td></tr><tr><td></td><td></td><td></td><td> Solução para escavação tecido cerebralestion </td></tr><tr><td> HBSS </td><td> Life Technologies </td><td> 14170-088 </td><td></td></tr><tr><td> A tripsina 2,5% </td><td> Life Technologies </td><td> 15090-046 </td><td> A concentração final: 0,25% </td></tr><tr><td></td><td></td><td></td><td> Outro </td></tr><tr><td> Etanol a 70% (vol / vol) </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> Água </td><td> PAA </td><td> S15-012 </td><td> grau de cultura de células </td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td> grau de cultura de células </td></tr><tr><td> Tente 0,05%psin-EDTA </td><td> Life Technologies </td><td> 25300-062 </td><td></td></tr><tr><td> Filtro de 0,45 ^ M estéril </td><td> Músculo da coxa </td><td> 16555 </td><td></td></tr><tr><td> 3,5 centímetros placa de Petri </td><td> BD Falcon </td><td> 353001 </td><td></td></tr><tr><td> 15 ml tubo Falcon </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> Tubo Falcon de 50 ml </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> 75 centímetros 2 frasco de cultura de tecidos </td><td> BD Falcon </td><td> 353136 </td><td></td></tr><tr><td> Fórceps, bem </td><td> Dumont </td><td> 2-1032, 2-1033 </td><td> # 3c; # 5 </td></tr><tr><td> Fórceps ponta plana </td><td> KLS Martin </td><td> 12-120-11 </td><td></td></tr><tr><td> 13 centímetros tesouras cirúrgicas </td><td> Aesculap </td><td> BC-140-R </td><td></td></tr><tr><td> Estereomicroscópio </td><td> Leica </td><td></td><td> MZ7.5 </td></tr><tr><td> Estereomicroscópio Câmera + </td><td> Leica </td><td></td><td> MZ16F; DFC320 </td></tr><tr><td> Microscópio Câmera + </td><td> Zeiss, Canon </td><td></td><td> Primo Vert; PowerShot A650 IS </td></tr><tr><td> Centrifugador </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> Banho de água </td><td> Julabo </td><td></td><td> SW20, 37 ° C </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

Os astrócitos são um tipo de célula abundante no cérebro dos mamíferos, mas há ainda muito que aprender sobre as suas características moleculares e funcionais. Nos sistemas de cultura in vitro de células de astrócitos podem ser utilizados para estudar as funções biológicas destas células gliais em detalhe. Este protocolo de vídeo mostra como obter astrócitos puras através do isolamento e da cultura de células corticais mistas de filhotes de rato. O método baseia-se na ausência de neurónios viáveis ​​e a separação dos astrócitos, oligodendrócitos e microglia, os três principais populações de células da glia do sistema nervoso central, em cultura. Imagens representativas durante os primeiros dias de cultura demonstram a presença de uma população mista de células e indicam o ponto no tempo, quando os astrócitos tornam-se confluentes e deve ser separada da microglia e oligodendrócitos. Além disso, demonstramos a pureza e a morfologia dos astrócitos em cultura astrocítica utilizando colorações imunocitoquímicos para bem estabelecida erecentemente descritos marcadores de astrócitos. Este sistema de cultura pode ser facilmente usado para obter os astrócitos de rato puros e astrócitos meio condicionado para estudar vários aspectos da biologia do astrócito.

Após o isolamento do cérebro de rato completo (Figura 1A), o cerebelo e os bulbos olfativos tem que ser removido (Figura 1B). Os córtices são descascadas do tronco cerebral do rato (Figura 1C) e meninges do córtex individual (Figura 1D &#39;) são cuidadosamente removida (Figura 1E). Meninges são óbvias pelo sistema artéria meníngea e resultados de remoção incompleta em contaminação da cultura final do astrócito por célul...

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

Isolation and Culture of Mouse Cortical Astrocytes

Os astrócitos têm sido reconhecidos como células versáteis que participam em processos biológicos fundamentais, que são essenciais para o desenvolvimento do cérebro e função normais, e reparação do sistema nervoso central. Aqui apresenta-se um processo rápido de obtenção de culturas de astrócitos de rato puros para estudar a biologia desta classe importante de centrais células do sistema nervoso.

Isolamento e Cultura de astrócitos corticais de rato

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

90.6K visualizações.  University of Freiburg. Os astrócitos têm sido reconhecidos como células versáteis que participam em processos biológicos fundamentais, que são essenciais para o desenvolvimento do cérebro e função normais, e reparação do sistema nervoso central. Aqui apresenta-se um processo rápido de obtenção de culturas de astrócitos de rato puros para estudar a biologia desta classe importante de centrais células do sistema nervoso.

Vídeo: 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 e Cultura Primária de Neurônios do hipocampo do rato embrionárias e Cortical

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

Neurociência

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 baseado em isolamento e cultura de motoneurônios embrionárias de rato

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-cultura de neurônios de rato Primária Cortical e Glia

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

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

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

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

Isolamento e Cultura de neurônios do hipocampo de ratos pré-natal

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.

Isolamento e Cultura de Células Papila fungiforme Humanos Taste

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.

Modelagem astrocitoma Patogênese<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Usando Cortical astrócitos ou células-tronco neurais de condicional, Geneticamente Modificadas Ratos

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.

As diferenças sexuais no mouse do hipocampo astrócitos após<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.

Isolamento e Cultura de células estaminais adultas neurais do Subcallosal Zona Rato

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.

Diferenciação de células estaminais embrionárias de rato em Cortical interneurônio precursores

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.