Agradecemos Jonna Ellis para a assistência editorial. O projeto descrito foi apoiada por número Prêmio R01MH079751 (PI: F. Peruzzi) do Instituto Nacional de Saúde Mental. O conteúdo é da exclusiva responsabilidade dos autores e não representam necessariamente as opiniões oficiais do Instituto Nacional de Saúde Mental ou o National Institutes of Health.

1. Poli-D-lisina (PDL): Preparação <ol><li> Adicionar 5 ml de ddH2O estéril a 5 mg de PDL para se obter uma solução stock de 1 mg / ml. </li><li> Misture solução estoque pipetando várias vezes. </li><li> Use imediatamente ou guarde Poly-D-lisina solução a 2-8 ° C. </li></ol> 2. Poli-D-lisina (PDL): Revestimento de plástico pratos de cultura de células <ol><li> Dilui-se a solução de reserva com PDL estéril ddH2O para a concentração final de 10 ug / ml. </li><li> Pipetar solução suficiente para um prato 60 milímetros para cobrir a área de cultura de superfície (3 ml para um prato de 60 mm). </li><li> Balançar suavemente para assegurar mesmo revestimento da superfície da cultura. </li><li> Incubar as placas revestidas à temperatura ambiente (RT) durante a noite. </li><li> No dia seguinte, geralmente o dia de dissecção, remover a solução de poli-D-Lisina por aspiração e lavar brevemente com 3 ml de estéril DDQ 2 O. Repetir este passo. Após a segunda lavagem, remover a água completamente por aspiração. </li><li>As placas podem ser armazenadas a 4 ° C durante até três semanas. </li></ol> 3. Poli-D-lisina (PDL) e laminina: Preparação e revestimento de vidro de duas câmaras lâminas <ol><li> Mistura PDL (1 mg / ml) e laminina (1 mg / ml) soluções de reserva em estéril ddH2O para a concentração final de 10 e 5 ug / ml, respectivamente. </li><li> Pipetar solução suficiente em poços de um vidro de duas câmaras de deslizamento para cobrir a área da superfície de cultura (1 ml para cada poço de um vidro de 2 bem duas câmaras de slides). </li><li> Balançar suavemente para assegurar mesmo revestimento da superfície da cultura. </li><li> Incubar as placas revestidas a noite à RT. </li><li> No dia seguinte, remover a solução de revestimento de poli-D-Lisina-laminina através de aspiração e lava-se brevemente duas vezes com 1 ml de estéril DDQ 2 O. Após a segunda lavagem, remover a água completamente por aspiração. </li><li> Lâminas de câmara pode ser armazenada a 4 ° C durante até três semanas. </li></ol> Nota: Qualquer slide câmara de vidro podem ser revestidas following protocolo esta. Muitas vezes usamos as lâminas duas câmaras porque cada slide fornece a configuração de controle de teste experimental (por exemplo, sem tratamento contra tratada, não transfectadas contra transfectadas). 4. Dissecção Neuronal e Cultura <ol><li> Aquecer os seguintes reagentes em um banho de água 37 ° C: <ul type="disc"><li> TrypLE Express no seu original garrafa de 100 ml. </li><li> Neurobasal/B27 médio completo (ver tabela I). O volume aquecida depende do número de pratos para ser revestido (30 ml por exemplo, para dez pratos de 60 mm banhados). </li></ul></li><li> Adicionar 3 ml de solução fria E o Hibernate para quatro pratos de 60 mm de cultura e 13 ml para uma BD de 15 ml Falcon tubo de polipropileno de alta claridade cônica. </li><li> Adicionar 25-30 ml de meio de dissecção fria (ver Tabela II, o Dr. Olimpia Meucci, comunicação pessoal) para cada um de três pratos de cultura de 100 mm. Estas placas, contendo um grande volume de meio, será usado para lavar os embriões imediatamente após a suaremoção das bolsas amnióticas (passos 4.7 e 4.8). </li><li> Euthanize um E17 rato timed grávida de CO 2 em conformidade com a Política Pública de Serviços de Saúde sobre a Atenção Humanizada e Uso de Animais de Laboratório e sob um cuidado animal institucional aprovado e usar o protocolo. </li><li> Pulverizar abdómen inferior com EtOH a 70% e cortar medialmente através da pele e dos músculos com um par de tesouras expondo o útero e embriões. </li><li> Remover todos os fetos e colocá-los em um prato de 100 mm estéril contendo um excesso de meio de dissecção fria (25-30 ml, ver passo 4.3). </li><li> Corte embriões usando uma tesoura pequena de bolsa amniótica e colocá-los no prato de 100 mm contendo meio segundo dissecção fria. </li><li> Lavar os embriões à temperatura ambiente por suavemente inclinação prato a 100 mm durante 5-10 segundos. Em seguida, transferir os embriões enxaguado para o prato milímetros terceiro 100 contendo meio de dissecação. Duas lavagens em meio de excesso são geralmente suficiente para remover todos os vestígios de sangue. No entanto, se nesário, lavar uma vez mais utilizando um prato de 100 mm fresco contendo 25-30 ml de meio de dissecção a frio. </li><li> Usando uma pinça estereomicroscópio e curvo, extrair cérebro de cada embrião rato puxando a pele e crânio. Coloque cérebro inteiro em um dos pratos de 60 mm (tipicamente, com não mais de 5 cérebros por placa) com frio Hibernate E. Mantenha estas placas em gelo. </li><li> Tomar um prato de cada vez e, sob um microscópio de dissecação, separar os hemisférios e isolar os córtices cerebrais remoção do mesencéfalo e as meninges. </li><li> Opcional: cérebros de corte ao longo da linha média, extrair hipocampos, e siga o procedimento abaixo para isolar os neurônios do hipocampo. </li><li> Colete todos os córtices dissecados em 15 ml tubo transparente cônico contendo 13 ml de frio Hibernate E. Deixe o córtex cerebral sobre o gelo até que todas as dissecções são concluídas. Devido ao seu pequeno tamanho, hipocampos dissecados podem ser recolhidos num tubo de Eppendorf de 1,5 ml em vez de um tubo de 15 ml. Se desejado, a este passo córtices ouhipocampos pode ser colocada num frasco criotubo contendo 1 ml de Hibernate E + 2% B27 + gentamicina (50 ug / ml) + Fungizone (250 ng / ml) na proporção de 2-4 córtices ou 2-4 hipocampos por frasco. O tecido cerebral pode ser armazenada a 4 ° C no escuro, durante até uma semana (mais tarde vezes não têm a ser testado ainda). Quando necessário, usar fórceps finos para transferir o tecido do cérebro para um tubo de 15 ml contendo Hibernate E e, em seguida, seguir o protocolo a seguir para isolar os neurónios. </li><li> Transferir o tubo para um capuz de cultura de tecidos. Permitir que os córtices para assentar no fundo do tubo e, então, cuidadosamente remover o sobrenadante. </li><li> Adicionar 13 ml de E Hibernate fresco para o tubo de 15 ml cónico, permitir córtices assentar no fundo do tubo e cuidadosamente remover o sobrenadante. Repita este passo mais 2 vezes e, após a última lavagem, remover cuidadosamente todas as mídias. </li><li> Enzimaticamente digerir córtices cerebrais por adição de 1-2 ml (dependendo do número de córtices; usar menos para o isolamento hipocampos) de Tr quentesypLE Express. Selar a tampa do tubo com Parafilm e flutuar o tubo num banho de água 37 ° C durante 10 minutos. </li><li> Pulverizar o tubo com etanol a 70% antes de abrir a tampa e adicionar 10 ml de Hibernate E. Permitir os córtices para assentar no fundo do tubo e remover o sobrenadante. Repita este passo três vezes para lavar TrypLE Express. Na última etapa, remova cuidadosamente todos os meios de comunicação. </li><li> Suavemente triturar (4-5 vezes) os córtices em 2 ml de meio completo utilizando Neurobasal/B27 um incêndio-polido vidro Pasteur (aproximadamente 1 mm de diâmetro). Tenha cuidado para evitar bolhas. </li><li> Repita mais 4-5 vezes com uma pipeta de Pasteur estéril de vidro menor em diâmetro (ou seja, uma pipeta de cerca de milímetros de diâmetro 1/2-3/4). Não use uma pipeta Pasteur menor do que isso, ou ele vai destruir as células. </li><li> Permitir que as restantes peças de tecido (em geral, muito poucos, se houver) para resolver. </li><li> Transferir a suspensão de célula única superior a um tubo de 15 ml-nova, deixando para trás peças estabelecidos de tecido. Pelether diluir a suspensão de células até 10-12 ml com Neurobasal/B27 meio completo. </li><li> Misturar bem e diluir as células para a contagem por adição de 10 ul de suspensão de células a 490 ul de solução de contagem de 50x (ver tabela III) em um tubo de Eppendorf de 1,5 ml. </li><li> Células da placa sobre placas revestidas PDL na densidade de 5,0 x 10 4 cm / 2. Se nucleofection é para ser realizado, recomendamos plaqueamento das células em uma concentração mais elevada (8-10 x 10 4 cm / 2). </li><li> Normalmente, nós dissecar 9-10 fetos por experiência, como cerca de 13 x 10 6 neurônios são derivados de cada feto E17. Se os embriões são necessários mais, certifique-se que o procedimento todo não durar mais de duas horas. </li><li> Se desejado, 24 horas após o isolamento, 10 mM de citosina-β-D-arabinofuranoside (AraC) pode ser adicionado a cada prato, a fim de impedir a proliferação glial. No entanto, este passo não é necessário uma vez que Neurobasal/B27 médio inibe a proliferação glial, Conforme as recomendações do fabricante (Invitrogen / Gibco). </li><li> Neurónios podem ser utilizados para experiências após 4-5 dias in vitro, embora o tempo exacto depende estágio de diferenciação desejado. Temos culturas de neurónios por até 4 semanas sem uma diminuição significativa na sobrevivência (Figura 1). </li><li> Para a cultura de estendida, substituir meio de cultura a cada semana com meio preparado de fresco Neurobasal/B27 completa. </li></ol> 5. Os resultados representativos Neurónios cultivados em lâminas câmara de vidro pode ser submetido a imunocitoquímica. A Figura 1 mostra uma imagem típica de um neurónio cortical fixado após cinco dias em cultura e immunolabeled com anti-MAP-2 anticorpo para mostrar os processos neuronais. A Figura 2 mostra uma imagem representativa de um neurónio de rato hipocampal após 3 semanas em cultura. A morfologia neuronal de uma célula diferenciada totalmente é realçada por MAP-2 immunolabeling (MAP-2 marcador neuronal, rato clone de anticorpo monoclonal AP-20, Gene Tex, Irvine, CA), seguindo um procedimento padrão como descrito anteriormente 1. As imagens foram visualizadas com o Eclipse Nikon E400 microscópio vertical de fluorescência equipado com câmera aquática EXI (Qimaging), motorizado eixo Z, e SlideBook5 aquisição / deconvolução software (Intelligent imagem Innovations, Inc., Denver, CO). Uma série de imagens tridimensionais de cada imagem individual foram deconvoluídos para uma imagem bidimensional e resolvidas, ajustando o sinal de corte com a intensidade de máximo próximo para aumentar a resolução. A Figura 3 mostra pureza das culturas neuronais. Lisados ​​de proteínas foram obtidos a partir de DIV7 culturas neuronais de rato (CTX) ea partir de um caso de glioblastoma humano (GBM). Como esperado, o lisado neuronal é fortemente positiva para a proteína neuronal MAP-2 e negativo para a GFAP marcador astrocítica; enquanto o lisado de proteína GBM é f negativoou MAP-2 e positiva para GFAP. Embora em nosso protocolo que temos vindo a utilizar Hibernate E por vários anos como dissecar e lavagem médio, recentemente, têm explorado o uso adicional e muito prático de se preservar tecidos cerebrais para uso posterior. A Figura 4 ilustra alguns dias in vitro 5 (DIV5) cultura de neurónios corticais de rato isolado a partir de córtices mantidos a 4 ° C durante uma semana em Hibernate E + B27 após a sua dissecção original a partir dos embriões. Os neurónios foram plaqueados em um copo de duas câmaras lâmina revestida com PDL e laminina, como descrito anteriormente. A imagem adquirida foi deconvoluídos usando SlideBook5 aquisição / software desconvolução como descrito acima (Figura 2). <img alt="A Figura 1" src="/files/ftp_upload/3965/3965fig1.jpg" /> Figura 1. Imagem representativas de um neurónio cortical nucleofected com pmaxGFP (Amaxa, Lonza, Walkersville, MD) e immunolabeled com MAP-2 Anticorpo, em vermelho. Aumento original de 100x. <img alt="A Figura 2" src="/files/ftp_upload/3965/3965fig2.jpg" /> Figura 2. Imagem representativas mostrando MAP-2 imunomarcação, em vermelho, de neurónios do hipocampo após 3 semanas em cultura. Coloração DAPI, em azul, mostra núcleos celulares. Aumento original de 40x. <img alt="A Figura 3" src="/files/ftp_upload/3965/3965fig3.jpg" /> Figura 3. Western blot mostrando pureza das culturas de células neuronais. 30 ug de rato lisados ​​de proteínas neuronais e humana GBM foram separados por electroforese e submetido a análise de Western blot seguindo procedimentos padrão 1. Anti-MAP-2 era um anticorpo policlonal de coelho a partir de sinalização celular (Danvers, MA), anti-GFAP anticorpo foi um monoclonal de ratinho da Chemicon (Millipore, Billerica, MA), e do rato anticorpo monoclonal anti-GRB2 era de Laboratories Transdução BD ( MD, faíscas). GRB2 foi utilizado como um controlo de carga. </p&gt; <img alt="A Figura 4" src="/files/ftp_upload/3965/3965fig4.jpg" /> A Figura 4. Imagens representativos de 5 dias in vitro (DIV5) neurónios corticais de ratos obtidos a partir de córtices deixadas no Hibernate E + B27 a 4 ° C durante uma semana após a sua dissecção. A) O contraste de fase de neurónios cultivados sobre um vidro de duas câmaras de slides. Aumento original 20X. B) imunofluorescência mostrando a expressão de MAP-2 em processos neuronais, em verde, a cultura foi negativo para a GFAP astrocitária marcador. Coloração DAPI, em azul, indica núcleos celulares. Aumento original de 40x.

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O método de dissecção e cultura de neurónios do hipocampo de ratos e cortical aqui descrito permite a realização de experiências usando quase puros culturas neuronais cultivadas num meio quimicamente definido (Figura 3). Embora os protocolos para os neurónios de cultura quase puros em meios isentos de soro foram anteriormente descritos 2,3,4, há alterações importantes feitas em nosso método. Diferente de protocolos tradicionais (isto é, Banker et al.) 5, temos substitu...

<table border="1"><tbody><tr><td> Reagente </td><td> Concentração </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> Quadro I. Neurobasal/B27 meio completo. <table border="1"><tbody><tr><td> Reagente </td><td> Concentração </td></tr><tr><td> Glicose </td><td> 16 mM </td></tr><tr><td> Sacarose </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 ug / 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> Osmolaridade </td><td> 320-330 mOsm </td></tr></tbody></table> Tabela II. Meio de dissecção. <table border="1"><tbody><tr><td> Reagente </td><td> Volume (uL) </td></tr><tr><td> Neurobasal/B27 meio completo </td><td> 240 </td></tr><tr><td> Trypan azul 0,4% </td><td> 250 </td></tr><tr><td> Total </td><td> 490 </td></tr></tbody></table> Tabela III. 50x Contando solução. <table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Cat. número </td></tr><tr><td> E Hibernate </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> De sulfato de gentamicina </td><td> Sigma Aldrich </td><td> G1264 </td></tr><tr><td> Glutamax mM 200 </td><td> Gibco, Invitrogen </td><td> 35050 </td></tr><tr><td> TrypLE expresso vermelho w / o fenol </td><td> Gibco, Invitrogen </td><td> 12604 </td></tr><tr><td> Citosina-β-D-arabinofuranoside cloridrato </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> A 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 azul 0,4% </td><td> Gibco, Invitrogen </td><td> 15250 </td></tr></tbody></table&gt; Tabela IV. Reagentes específicos. <table border="1"><tbody><tr><td> Equipamento </td><td> Companhia </td><td> Cat. número </td></tr><tr><td> Microscópio estéreo </td><td> Olimpo </td><td> SZ61 </td></tr><tr><td> Pinças grandes </td><td> FST </td><td> 11022-14 </td></tr><tr><td> Pinça de ponta fina </td><td> Moria </td><td> MC40B </td></tr><tr><td> Micro pinça de ponta fina </td><td> Moria </td><td> MC31 </td></tr><tr><td> Afiadas tesouras </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> Micro tesoura de dissecção </td><td> FST </td><td> 91460-11 </td></tr><tr><td> Dissecando Micro tesoura curva </td><td> FST </td><td> 14067-11 </td></tr><tr><td> Vidro 2-câmara lâminas </td><td> Lab-Tek </td><td> 154461 </td></tr><tr><td> 60 pratos 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 frasco crio-tubo </td><td> Nunc </td><td> 375353 </td></tr></tbody></table> Equipamentos Quadro V. específico.

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

Estamos descrevendo um método rápido para dissociar cultura e os neurônios do hipocampo ou cortical a partir de embriões de ratos E15-17. O procedimento pode ser aplicado com sucesso para o isolamento de rato e humanos neurónios primários e progenitores neurais. Neurónios dissociadas são mantidas em meio isento de soro até várias semanas. Estas culturas podem ser utilizados para nucleofection, imunocitoquímica, a preparação de ácidos nucleicos, bem como electrofisiologia. Mais velhos culturas neuronais também podem ser transfectadas com uma taxa de uma boa eficiência de transdução por lentivirus e, de forma menos eficiente, com fosfato de cálcio ou de lípidos métodos baseados em tais como lipofectamina.

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

Nós descrevemos uma metodologia rápida para isolar e cultivar os neurônios do hipocampo e cortical a partir de embriões de roedores. Este protocolo permite a realização de experimentos em que quase puras culturas neuronais são necessários.

Isolamento e cultivo de células neurais embrionárias de ratos: um 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 ...

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38.0K visualizações.  LSU Health Sciences Center - New Orleans. Nós descrevemos uma metodologia rápida para isolar e cultivar os neurônios do hipocampo e cortical a partir de embriões de roedores. Este protocolo permite a realização de experimentos em que quase puras culturas neuronais são necessários.

Vídeo: 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.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

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

Neurociência

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.

Isolamento e Expansão do Rato Adulto células-tronco neurais utilizando o ensaio Neurosphere

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.

Estabelecer embrionárias de rato Cultura de Células-tronco neurais utilizando o ensaio Neurosphere

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.

Isolamento e Cultura de células da crista neural-tronco de folículos capilares humanos

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 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. ...

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 ...

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.

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

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.

Dissecção, Cultura, Análise e de<em&gt; Xenopus laevis</em&gt; Tecido embrionário da retina

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.

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

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.

Um rato, duas culturas: isolamento e cultivo de células-tronco neurais Adulto de as duas zonas neurogênica Individual Ratos

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