Os autores gostaria de agradecer a Christine Auste o seu apoio com a gravação de vídeo e Chalid Ghadban por sua excelente assistência técnica. Urszula Grabiec foi apoiada pelo Roux Programm FKZ 29/18.

experiências em animais foram realizadas em conformidade com a política da ética e da política sobre a utilização de animais na investigação neurociência como aprovado pela Europeu comunidades Conselho Directiva 2010/63/UE do Parlamento Europeu e do Conselho da União Europeia relativa à protecção dos animais utilizados para fins científicos.
 1. preparação dos instrumentos e meios de cultura <ol><li>para a preparação de OHSC usar o seguinte conjunto de instrumentos: duas tesouras pequenas, duas pinças curvas, uma pinça com ponta fina, três lâminas (duas de tamanho 11, um de tamanho 15), três suportes de bisturi, ronda os papéis de filtro (diâmetro: 35mm), agar, uma lâmina de barbear, um não modificado pipeta Pasteur, e um modificado pipeta Pasteur sem uma dica. Esterilize todos os materiais em autoclave antes do uso ( Figura 1).</li>
	<li>Pesa agar 5g e dissolvê-lo em 100 mL de água destilada. Esterilize a solução por 20 min a 121,7 ° C, a 210.8 kPa em autoclave. Distribuir a 3 mL de solução de ágar líquido em placas de Petri 60mm usando uma pipeta de vidro estéril, deixe-o solidificar por 5h, cubra com filme plástico parafina para evitar contaminação e armazenar a 4 ° C até nova utilização. Blocos de ágar são necessários para estabilizar o cérebro durante o procedimento de corte.</li>
	<li>Media <ol><li>fazer 200 mL do meio de preparação (pH 7,35) consistindo de meio essencial mínimo (MEM) de 198 mL e 2 mL de solução de L-glutamina (concentração final de 2 milímetros). Preparar a solução no dia da preparação de mídia e armazená-lo em 4 ° C.</li>
		<li>Preparar 100 mL de meio de cultura composto por 49 mL MEM, 25ml Hanks &#39; equilibrada soluções salinas (HBSS), soro de cavalo normal de 25 mL (v/v) (NHS), 1 mL de solução de L-glutamina (concentração final 2 mM), 100 µ g insulina, glicose de 120 mg, estreptomicina 10 mg , 10.000 U de penicilina e 800 µ g de vitamina C, como relatado anteriormente. Aquecer o meio (37 ° C), ajuste o valor de pH para filtro pH 7,4 e estéril (tamanho de poro de 0,2 µm). Repita o procedimento (aquecimento, ajuste de pH, etc.) em dois dias antes de mudar o meio. Utilize o meio, no máximo, por uma semana quando conservado a 4 ° C.</li>
	</ol></li> <li>Encha uma 35mm placa de Petri com meio de preparação para armazenar o cérebro. Coloque dois pratos de Petri vazia para coletar o tecido em um bloco de arrefecimento na área de trabalho.</li>
</ol> 2. Preparação e fatiar com uma Vibratome <ol><li>uso cérebros de ratos de 7-9 dias de idade ou 4 a 5 dias velhos ratos para a preparação de OHSC de acordo com Stoppini et al. 4 após a decapitação de animais, retire a pele do crânio com uma tesoura.</li>
	<li>Introduzir a lâmina de uma tesoura bem o buraco occipital e abrir o crânio por corte ao longo do eixo (dianteiro) (volta) rostral caudal. Fazer dois cortes perpendiculares ao primeiro, para que a tesoura aponta para a orelha esquerda e direita, respectivamente (&#34; T-incisão &#34;).</li>
	<li>Abrir o crânio cuidadosamente com a pinça fina, prestando atenção para não machucar o cérebro. Usar um bisturi (tamanho da lâmina 11) para cortar a parte mais rostral do polo frontal e o cerebelo.</li>
	<li>Por meio de uma espátula, retire o cérebro e coloque-o cuidadosamente na caixa de Petri preenchida com meio de preparação ( Figura 2). Posicione o cérebro sobre o suporte de amostra e corrigi-lo com cola de cianoacrilato médica. Usar as peças do ágar para assegurar a estabilização mecânica.</li>
	<li>Dissecar o tecido horizontalmente em 350 µm espessura OHSC usando um deslizamento vibratome.</li>
	<li>Avaliar as fatias opticamente, usando um microscópio binocular. OHSC de baixa qualidade descarte imediatamente. É importante tomar somente aquelas fatias com cytoarchitecture intacto, isolado da parte média do hipocampo (ver figuras 3 e 4) entre a dorsal e ventral hipocampo.</li>
	<li>Separar a região do hipocampo e córtex entorhinal usando um bisturi (redondo, tamanho da lâmina 15; a Figura 3). Deve ser preservado o córtex de percurso e entorhinal perforant.</li>
	<li>OHSC de seis a oito são obtidos de cada cérebro. 2-3 fatias de transferência para inserção de cultura de uma célula (poros tamanho 0.4 µm) e colocá-lo em um poço de um prato de cultura 6-poços contendo 1 mL de meio de cultura por alvéolo.</li>
	<li>Incubar os pratos 6-poços em 35 ° C em uma atmosfera totalmente umidificada com 5% (v/v) de CO 2 e mudar o meio de cultura celular cada segundo dia. 
	Nota: Realizar suas experiências no dia 6 em vitro (div). Reações inflamatórias associadas com o fatiamento procedimento desaparecem por dia 6. Nesta fase, as células de microglial apresentam uma morfologia ramificam novamente e conexões sinápticas amadureceram.</li>
</ol> 3. Avaliação da qualidade do tecido <ol><li>fixação, rotulagem e visualização de neurônios degeneram em OHSC <ol><li>após a realização dos experimentos, incubar o OHSC com iodeto de propidium 5 µ g/mL (PI) por 2 h antes da fixação, em ordem para manchar os núcleos dos neurônios degeneram. 
		Atenção: O PI é um suspeito cancerígeno, use sempre equipamentos de proteção individual (EPI) tais como luvas.</li>
		<li>Lavar o OHSC com tampão de fosfato 0,1 M (pH 7,4) e corrigi-los com uma solução de 4% (v/v) de paraformaldeído (PFA) por 24 h. 
		Cuidado: PFA é um carcinógeno tóxico e suspeito. Trabalhar sob a coifa e usar EPI.</li>
		<li>Lavar o OHSC em inserções com 1 mL de PBS e use um pincel de rigger para separar as fatias da membrana.</li>
		<li>Rotular o OHSC com tintura de IB 4 em uma placa de 24 ( Figura 5).</li>
	</ol></li> <li>Condução de experimentos de invasão do tumor usando fluorescente etiquetado células únicas <ol><li>24 h antes do início do experimento, rotular as células do tumor, usando o tintura fluorescente Carboxyfluorescein diacetato grufo éster (SE CFDA).</li>
		<li>Desanexar e contar as células de tumor utilizando uma câmara de Neubauer.</li>
		<li>Ressuspender as células em meio, tal que 10 µ l de suspensão contém o número de celular desejado (normalmente 50.000 ou 100.000 células).</li>
		<li>Aplicar 10 µ l de suspensão de célula para a cultura de fatia e permitir que as células para invadir por 3 dias.</li>
		<li>No final do experimento, corrigir as culturas fatia usando 4% (v/v) PFA para 24h e rotular as culturas co com PI para outro 24h para visualizar o cytoarchitecture. 
		Cuidado: PFA é um carcinógeno tóxico e suspeito. Trabalhar sob a coifa e usar EPI.</li>
		<li>Montar as culturas co em uma lamela para posterior análise utilizando o meio de montagem.</li>
	</ol></li></ol> 4. Avaliação das experiências de OHSC analisar o OHSC com um laser confocal microscópio (CLSM). Para a deteção de PI rotulado, degenerando neurônios ou o PI rotulado cytoarchitecture usar a luz monocromática do comprimento de onda λ = 543 nm e uma banda de emissão passam filtro para comprimentos de onda λ = 585-615 nm. Para o CFDA rotulado de células tumorais ou IB 4 micróglia, usar um comprimento de onda de excitação de λ = 488 nm. Para ambos os tipos experimentais, gravar uma z-pilha com µm 2 fatias grossas de óptica e usado para a avaliação.

Os autores não têm nada a divulgar

O presente protocolo descreve a preparação de OHSC. Esse modelo permite que o teste de recursos intrínsecos e reações do tecido cerebral após a aplicação de estímulos fisiológicos e patológicos. Além de análises de parâmetros eletrofisiológicos, OHSC pode ser lesado e os efeitos de dano em todos os tipos de células podem ser determinados. Tratamento com substâncias diferentes e a descrição detalhada dos processos de maciços ou na ausência de macrófagos e linfócitos de cura é possível.
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OHSC é um modelo bem caracterizadas em vitro para estudar propriedades fisiológicas e patológicas dos neurônios, astrócitos e microglia1. É fácil de controlar o ambiente extracelular e monitorar as mudanças morfológicas e celulares após vários estímulos. A organização dos neurônios hippocampal e suas conexões estão bem conservadas após preparação2,3. Fora várias vantagens, OHSC permitem o monitoramento da invasão de lesão e tumor cerebral sem cirurgia animal. Seis a oito OHSC pode ser obtido de um único cérebro de roedor. OHSC, portanto, ajudar a significativamente reduzir o número de animais e permitir testes várias concentrações de droga, manipulações genéticas ou modelos de lesão diferentes no mesmo animal. Fatia com base em ensaios, em condições experimentais podem ser precisamente controladas. Além disso, desenvolvimento dependente do tempo de condições patológicas como danos secundários facilmente pode ser monitorado pela imagem latente de lapso de tempo.
No protocolo determinado, originalmente estabelecido por Stoppini et al 4, as etapas de preparação são descritas e destacam-se importantes marcos morfológicos para a seleção de fatias apropriadas. Recomendamos a preparação de pós-Natal dia 7-9 ratos ou camundongos pós-Natal dia 4-5. Nesses períodos, OHSC mostram uma resistência robusta de traumas mecânicos e um elevado potencial para a reorganização dos circuitos neuronais. Em contraste, preparações de ratos embrionários ou adultos rapidamente mudam sua estrutura e perdem sua morfologia de organotypic durante o cultivo e, portanto, são menos adequadas para o estudo de processos a longo prazo em investigação básica5,6 , 7 , 8 , 9 , 10 , 11. outro ponto crítico para a taxa de sobrevivência de OHSC é a espessura da fatia em si, como a difusão e, portanto, o fornecimento de nutrientes são limitadas12,13,14.

<table><tbody><tr><td>6-Well</td><td>Falcon</td><td>35-3046</td><td></td></tr><tr><td>Agar</td><td>Fluka</td><td>5040</td><td></td></tr><tr><td>Autoclav</td><td>Systec</td><td>DX-45</td><td></td></tr><tr><td>CFDA</td><td>&amp;nbsp;Thermo Fisher</td><td>V12883</td><td></td></tr><tr><td>Confocal laser scanning microscope (CLSM) LSM700</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Eagle&amp;acute;s Minimal Essential Medium&amp;nbsp;</td><td>Invitrogen</td><td>32360-034</td><td></td></tr><tr><td>Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I</td><td>Vector Labs</td><td>FL-1101</td><td></td></tr><tr><td>Glucose</td><td>Merk</td><td>1083371000</td><td></td></tr><tr><td>Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>Hank&amp;acute;s Balanced Salt Solution (with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Invitrogen</td><td>24020-133</td><td></td></tr><tr><td>Hank&amp;acute;s Balanced Salt Solution (without Ca&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Invitrogen</td><td>14170-138</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>I5500</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma Aldrich</td><td>A5960</td><td></td></tr><tr><td>L-Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>LN229</td><td>Cell-Lines-Service</td><td>300363</td><td></td></tr><tr><td>Medical cyanoacrylate glue (Histoacryl glue)</td><td>B.Braun</td><td>1050052</td><td></td></tr><tr><td>Millicell Culture Inserts</td><td>Millipore</td><td>PICMORG50</td><td></td></tr><tr><td>NMDA N-methyl-D-aspartic acid</td><td>Sigma Aldrich</td><td>M3262</td><td></td></tr><tr><td>Normal Horse Seum</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Petri dishes (all sizes)</td><td>Greiner</td><td>627160/664160/628160</td><td></td></tr><tr><td>PFA</td><td>Roth</td><td>0335.1</td><td>toxic</td></tr><tr><td>Propidium iodid (PI)</td><td>Sigma Aldrich</td><td>81845-25MG</td><td>toxic</td></tr><tr><td>U138</td><td>ATCC</td><td>HTB-14</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>Leica VT 1200</td><td></td></tr></tbody></table>

organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells

Em culturas hippocampal fatia de organotypic (OHSC), as características morfológicas e funcionais dos neurônios e células gliais estão bem conservadas. Este modelo é apropriado para abordar questões de pesquisa diferentes que envolvem estudos na neuroproteção, eletrofisiológicos experiências em neurônios, redes neuronais ou invasão do tumor. A arquitetura hippocampal e atividade neuronal em circuitos multisynaptic estão bem conservados em OHSC, mesmo que o próprio procedimento corte inicialmente lesões e leva à formação de uma cicatriz glial. A formação de cicatriz presumivelmente altera as propriedades mecânicas e comportamento difusiva de pequenas moléculas, etc. Fatias de permitam o monitoramento de processos dependentes de tempo após a lesão cerebral sem cirurgia animal e estudos sobre interações entre vários tipos de células derivado do cérebro, ou seja, astrócitos, micróglia e neurônios sob fisiológicos e patológicos condições. Um aspecto ambivalente deste modelo é a ausência de fluxo sanguíneo e as células imunes do sangue. Durante a progressão da lesão neuronal, migração de células do sistema imunológico da peça sangue um papel importante. Como essas células estão falta em fatias, os processos intrínsecos da cultura podem ser observados sem interferência externa. Além disso, em OHSC a composição do ambiente externo médio é precisamente controlada. Uma vantagem adicional desse método é o menor número de animais sacrificados, em comparação com preparações padrão. Vários OHSC pode ser obtido de um animal, possibilitando estudos simultâneos com vários tratamentos em um animal. Por estas razões, OHSC são adequados para analisar os efeitos das novas terapias protetora após dano tecidual ou durante a invasão do tumor.
O protocolo apresentado aqui descreve um método de preparação de OHSC que permite gerar altamente reprodutível, bem preservado fatias que podem ser usadas para uma variedade de pesquisa experimental, como estudos de invasão de neuroproteção ou tumor.

Estudos de neuroproteção: Para determinar o dano neuronal, o número de núcleos positivos de PI e IB4 positivo microglia em cada terceira secção óptica da camada de células grânulo (GCL) de giro do pectínea (DG) foi contado. Para experiências de invasão do tumor, o z-projeção de intensidade máxima da pilha foi usado para calcular a área coberta por células tumorais, como uma medida de invasão e Visualizar padrões diferentes de invasão (<stron...

Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Culturas de fatias hippocampal organotypic (OHSC) representam um modelo in vitro que simula a situação na vivo muito bem. Aqui descrevemos uma vibratome de base melhorada fatiar protocolo para obter fatias de alta qualidade para uso em avaliar o potencial neuroprotetor do romance substâncias ou o comportamento biológico das células tumorais.

Organotypic fatias Hippocampal culturas como um modelo para estudo neuroproteção e invasão de células tumorais

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

organotypic-hippocampal-slice-cultures-as-model-to-study

Research

JoVE Journal

Neuroscience

12.8K visualizações.  Martin Luther University Halle-Wittenberg. Culturas de fatias hippocampal organotypic (OHSC) representam um modelo in vitro que simula a situação na vivo muito bem. Aqui descrevemos uma vibratome de base melhorada fatiar protocolo para obter fatias de alta qualidade para uso em avaliar o potencial neuroprotetor do romance substâncias ou o comportamento biológico das células tumorais.

Vídeo: Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Cultures Organotípicas fatia de Estudos de Pós-natal Neurogênese

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Biologia do Desenvolvimento

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

O modelo de Cultura organotípicas Hippocampal Slice para o exame das lesões neuronais

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Neurociência

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotípicas Hippocampal Culturas Slice

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Um modelo de fatia cerebral ex vivo para estudar e direcionar o crescimento do tumor metastático do câncer de mama

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Pesquisa do câncer

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Sistema de co-cultura com uma fatia organotípicas Cérebro e esferóide 3D de carcinomas de células

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Medicina

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

A Análise de Neurovascular Remodelação em Entorhino-hipocampal culturas Organotípicas Fatia

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Um tumor cerebral / Slice organotípicas Co-cultura Sistema de Estudos de Tumor Microenvironment e alvejado droga Terapias

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Um modelo de cultura de fatia organotipica de glioblastoma humano para estudo de migração de células tumorais e efeitos específicos de pacientes de drogas anti-invasivas

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Um Modelo de Epipetogênese em Culturas de Fatia Organotipica Do Cortex-Hipocampo Rhinal

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Source: Chadwick, E. J. et al. <a href="https://app.jove.com/v/53304">A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.</a> J. Vis. Exp. (2015).
This video demonstrates the development of a 3D co-culture system. Mouse brain slices are placed on a membrane, and fluorescently labeled human brain tumor cells are allowed to grow on it. Once the system is ready, it is stained for imaging.

Estabelecendo um tumor cerebral em um modelo de co-cultura de fatia organotípica

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plating the Slices onto the ...