Esta pesquisa foi suportada por concessões do Programa de Graduação Oportunidades de Pesquisa da UCI (AR e SP) e subvenções do Estado da Califórnia Initiative da doença de Alzheimer e os Institutos Nacionais de Saúde não conceder. HD38466, e doença de Alzheimer Research Center não conceder. AG16573 (JB)

1. Criopreservação de tecido cortical Blocos Preparação de material <ol><li> Preparar a solução de 1X Hank Sal Buffered (HBSS), diluindo a solução estoque 10X em água estéril (1:9). HBSS armazenar a 4oC. </li><li> Prepare meio de congelamento de 10% DMSO por diluição de DMSO em HBSS (1:9). O meio de congelamento deve ser feita antes da criopreservação fresco e armazenado a 4oC até refrigerados. </li><li> Uma lâmina de barbear de aço é usada para o corte sistemático de tecido. Antes do uso, a lâmina de barbear é esterilizado por submersão em etanol 70% para 2 hr. Imediatamente antes da transformação do tecido, lavar a lâmina com água estéril 3 vezes. Evite deixar a lâmina em água estéril para uma quantidade excessiva de tempo, como é propensa à oxidação. </li><li> Um recipiente de congelamento Nalgene é carregado com criotubos (2,5 mL) e depois levados a um gabinete estéril biológica. 1 mL de meio de congelamento refrigerados é adicionado a cada frasco. O recipiente de congelamento é então colocada a 4oC por pelo menos 2 horas. </li></ol> Limpeza, Chopping, e Congelamento <ol><li> O tecido cerebral para ser congelado é limpo da membrana meníngea e vasos sanguíneos. Use dicas agulha estéril para remover cuidadosamente os restos de tecido, enquanto trabalhava em cima de um bloco de gelo. Tecido limpas devem ser lavadas levemente com HBSS frio e transferidos para uma placa de Petri nova para cortar. </li><li> Trabalhando em cima de um bloco de gelo, use uma lâmina de barbear esterilizados para rapidamente cortar o tecido em aproximadamente 1 mm a 3 quadras. Trabalhar com pequenas porções de tecido limpo em um tempo resulta em um melhor controle sobre o processo de corte, resultando em tamanhos de bloco mais uniforme. </li><li> Lentamente, adicionar o tecido cortado em 50 mL de HBSS em um tubo cônico de 50 mL. Lave o prato de Petri com HBSS a fim de recolher todos os blocos tecido remanescente. Permitir que os blocos de tecido para descer ao fundo do tubo, criando um tecido ligeiramente comprimido bloco pellet. </li><li> Enquanto o tecido está se instalando, trazer o recipiente previamente refrigerados congelamento e criotubos para o gabinete de segurança biológica. Destape todos os criotubos para apressar o processo de adição de tecido. </li><li> Depois de todos os blocos de tecido se instalaram, aspirar cuidadosamente a HBSS excesso, deixando uma camada muito fina de mídia acima da pelota. Centrifugação é desencorajado, pois ele fará com que os blocos de tecido para aderir uns aos outros, diminuindo significativamente a eficiência de congelamento. </li><li> Lentamente coletar 200 mL do fundo do bloco de tecido solto pellet usando ponteira com orifícios de largura (cortar a ponta com uma tesoura esterilizada). Transferir o material para um cryovial e passar para o tecido, ao lado de novo recolher do fundo do sedimento. É essencial que o procedimento de atribuição de todo o tecido não leva mais do que 3-4 min por recipiente de congelamento. Isso garante que o tecido é exposto a DMSO por apenas um curto período de tempo antes do congelamento. Depois de transferir o tecido para os frascos, colocar o recipiente em congelamento a-80oC freezer por pelo menos 4 horas. Alternativamente, o recipiente de congelamento podem ser deixados durante a noite a -80 oC. Repita esse processo para todos os recipientes de congelamento restantes. </li><li> Transferir os criotubos do recipiente de congelamento (s) para um cryobox e coloque em um tanque de nitrogênio líquido para armazenamento de longo prazo. </li></ol> 2. Descongelamento e Cultivo de Blocos Congelados tecido cortical Preparação de material <ol><li> Um dia antes de amostras de tecido de descongelamento, poli-L-lisina revestido Petri pratos / lamelas estão preparados. Em geral, para os neurônios corticais que preparam pratos revestido com 500 mcg / mL ou 1 mg / mL. Adicione o suficiente solução de poli-L-lisina (feita em tampão borato) para cobrir totalmente o fundo do prato. Se lamínulas de vidro são necessários, garantir que eles são completamente submerso na solução de poli-L-lisina. Incubar por pelo menos 12 hr. Antes de usar, lave os pratos com uma quantidade generosa de água estéril 3 vezes, 5 min cada. </li><li> HBSS quente é usado para limpar o tecido descongelado, bem como dissociar o tecido em células em suspensão. O volume de HBSS necessário irá variar dependendo da quantidade de tecido a ser descongelado, mas normalmente um cryovial irá diluir em 50 mL de HBSS e será dissociada em 10 mL de HBSS. </li><li> Modificado Dulbecco Medium Eagle com 10% de ferro suplementado bovinos soro bovino (EBS) e 1% antibiótico / antimicótico (AA) mistura (DMEM (++)) deve ser colocado em banho-maria a 37 ° C. </li><li> Médio chapeamento (NB (+++)) deve ser feito fresco antes de usar. Este meio é preparado pela adição de 1% antibiótico / antimicótico mais B27 e suplementos N2. </li></ol> Nota: Nomes de mídia anexados com cruzes (+) indicam a inclusão de aditivos à base de composição dos meios de comunicação. Neste texto, DMEM (+ +) denota DMEM acrescido de 10% EBS mais 1% AA, enquanto NB (+++) denota NB plnos 1% AA mais B27 além de N2. Descongelamento e Cultivo <ol><li> Remova a criotubos do tanque de nitrogênio líquido e descongelamento rápido a 37 ° C em banho-maria até que apenas uma pequena pelota de gelo é observada dentro do conteúdo do frasco. </li><li> Gentilmente transferir o conteúdo para frasco 50 ml de HBSS quente em um tubo cônico. Use uma ponta de orifício largo para desalojar qualquer gentilmente blocos tecido remanescente preso no cryovial. Inverter o tubo 3-4 vezes e permitir que o tecido para coletar lentamente na parte inferior. Tecido deve resolver rapidamente, mas se os blocos são muito pequenos isso não pode ocorrer e luz centrifugação (~ 200 x g) é recomendado. </li><li> Aspirar HBSS excesso. </li><li> Adicionar 10 mL de HBSS quente para o tecido da pelota e 300 mL de tripsina 0,25% e 50 mL de DNAase. Incubar em banho-maria a 37 ° C por 5 min, em seguida, coloque em um agitador orbital ajustado para 80 rpm e 37 ° C para um adicional de 5 min. </li><li> Após este período, trazer os blocos de tecido em um gabinete de segurança biológica e usar uma pipeta 10 mL com cuidado agitar os blocos de tecido até obter uma suspensão celular nublado é formado. Desactivar a tripsina, adicionando 10 mL de DMEM quente (+ +) para a suspensão de células. </li><li> Centrifugar as células dissociadas em 1200 x g por 5 min. Aspirar e desprezar o sobrenadante, ressuspender o pellet celular em 10 mL de DMEM quente (+ +) e quantificar o número de células usando um hemocitômetro. </li><li> Células placa de poli-L-lisina pratos revestido. Tipicamente, um prato de 60 mm será revestida com 1x10 6 células. </li><li> Permitir que as células para anexar ao prato / lamínulas durante aproximadamente 1 hora na cultura de tecidos incubadora. É recomendado para monitorar o progresso de apego em uma placa a cada 10 minutos até apego eficaz é observado. Então, gentilmente substituir o meio com DMEM fresco (++). </li><li> Após 24 horas, substituir o DMEM (+ +) com água morna NB (+++). Alterações médias parciais (50%) são realizadas a cada 5 dias com novas NB (+++). Culturas saudáveis ​​normalmente apresentam sinais de diferenciação e crescimento dos processos após 24 hrs. Sob essas condições, e realizando mudanças médio parcial a cada 4-5 dias, as culturas podem ser mantidas por períodos prolongados de tempo até 4-6 semanas. </li><li> Para a geração de células precursoras neuronais (NPCs), um protocolo semelhante é usado com a ressalva de que após a centrifugação, DMEM sem EBS é usado. As células são plaqueadas em frascos T-25 em DMEM/F12 (1:3), suplementado com B27 e EGF (20 ng / mL) para permitir a formação de neuropheres. Para diferenciar NPCs, neurospheres são semeadas em laminina (10 ug / mL) lamínulas revestido em DMEM/F12 (1:3) meio suplementado com N2. </li></ol> 3. Resultados representante Culturas saudáveis ​​irá mostrar um crescimento significativo e diferenciação após 5 dias pós-descongelamento e normalmente irá estabilizar em seu crescimento por 10 dias (Figura 1). Coloração Immuncocytochemical destas culturas revela numerosos astrócitos (Figura 2A) e neurônios (Figura 2B), tanto para humanos e ratos culturas primárias neuronais. Este protocolo também é adequado para a geração de NPCs como livre-flutuante neurospheres (Figura 3A), que, em condições de diferenciação, resultam em culturas de alta qualidade mista (Figura 3B, C). <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="Figura 1" /> Figura 1. Culturas de células de tecidos humanos congelados cortical. Congelados humana blocos de tecido cortical são descongeladas e semeadas em poli-lisina lamínulas revestido e cresceu por 10 dias. De dia 5 expansão celular é aparente e por 10 dias confluência seja alcançado. Barra de escala: 100 mm. <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="Figura 2" /> Figura 2. Coloração imunocitoquímica de culturas de células. (A) As culturas foram coradas com o marcador glial fibrilar glial proteína ácida (GFAP, setas grossas). (B) Os neurônios foram detectados com o marcador neuronal beta-tubulina III (TIII, setas finas). (C) Ambas as culturas humanas e de ratos mostram abundantes e glia neurônios após 10 dias de cultura. Anticorpos primários: mouse anti-GFAP (1:1000) e de coelho anti-TIII (1:1000). Anticorpos secundários: anti-rato Alexa 594 (1:500) e anti-coelho Alexa 488 (1:500). Coloração núcleos: Hoestch azul (1:1000). Barra de escala: 50 mm. <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="Figura 3" /> Figura 3. Geração de neurospheres. (A) de tecido congelado foi processado como descrito acima e permissão para se propagar como neurospheres. (B) Neurospheres foram semeadas em lamínulas em condições de diferenciação e crescimento por 10 dias, resultando em células migram longe do neurosphere. (C) Ambos os neurônios e astrócitos estão presentes na franja expansão de um neurosphere diferenciação. Contracoloração nuclear, os anticorpos primário e secundário usado como na Figura 2.

<ol>
	<li>Robbins, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+brain+tissue.">Cryopreservation of human brain tissue.</a> Exp Neurol. 107, 208-213 (1990).</li><li>Ware, C. B., Nelson, A. M., Blau, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled-rate+freezing+of+human+ES+cells.">Controlled-rate freezing of human ES cells.</a> Biotechniques. 38, 879-880 (2005).</li><li>Paynter, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practical+issues+for+cryopreservation+of+nerve+cells.">Principles and practical issues for cryopreservation of nerve cells.</a> Brain Res Bull. 75, 1-14 (2008).</li><li>Thirumala, S., Gimble, J. M., Devireddy, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+stromal+vascular+fraction+of+adipose+tissue+in+a+serum-free+freezing+medium.">Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.</a> J Tissue Eng Regen Med. 4, 224-232 (2010).</li></ol>

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Criopreservação oferece a oportunidade de amostras de cérebro banco precioso tecido para uso futuro. Aqui nós descrevemos um protocolo simples, mas eficaz para gerar tanto neurônio enriquecido culturas e células precursoras neuronais a partir de blocos de tecido congelado cérebro. Este procedimento evita os custos econômicos das técnicas de criopreservação tradicionais que utilizam mais caro taxa controlada freezers. O protocolo permite a geração de culturas viáveis ​​neuronal pós-descongelamento, proporcionando um meio rápido, mas eficaz para congelar blocos de tecido. O processo de congelamento completo pode demorar tão pouco como 20 minutos. Além de culturas primárias de neurônios, o uso desse blocos de tecido método também pode ser descongelado para gerar NPCs cresceu como livre neurospheres flutuante. A este respeito, a falta de soro no nosso meio de congelamento garante que as células são preservadas em um estado indiferenciado. Em condições de diferenciação, neurospheres produzido a partir de tecidos congelados de expansão show e as taxas de diferenciação muito comparável à neurospheres gerados a partir de tecido fresco.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995-073</td>
<td>High gluclose 1X</td>
</tr>
<tr>
<td>Bovine calf serum supplemented</td>
<td>&nbsp;</td>
<td>HYCLONE</td>
<td>SH30072.03</td>
<td>For culture medium </td>
</tr>
<tr>
<td>Neurobasal-A Medium (1X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>1088-022</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>N2 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>Trypsin (10X)</td>
<td>&nbsp;</td>
<td>Cellgro </td>
<td>25-054CI </td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Deoxyribonuclease 1 from bovine pancreas (DNase)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4527-30KU</td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) (10X)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14065</td>
<td>Cleaning tissue, washes, and freezing medium</td>
</tr>
<tr>
<td>Poly-L-Lysine- 500mg</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2636</td>
<td>Substrate for adhesion of neuronal cells</td>
</tr>
<tr>
<td>antibiotic-antimycotic (100X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>To prevent contamination</td>
</tr>
<tr>
<td>Large orifice pipette Tips (1-200 ul)</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>02-681-141</td>
<td>To prevent shear stress to cells</td>
</tr>
<tr>
<td>Graduated pipette tips (101-1000 ul)</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1111-2721</td>
<td>&nbsp;</td>
<tr>
<td>21 G1 precision guide needles</td>
<td>&nbsp;</td>
<td>Beckton Dickinson</td>
<td>305165</td>
<td>To clean tissue</td>
</tr>
<tr>
<td>10 ml pipette </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1071-0810</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>926-9-04</td>
<td>&nbsp;</td>
<tr>
<td>Single edge razor blade</td>
<td>&nbsp;</td>
<td>Smith Brand</td>
<td>67-0238</td>
<td>To chop tissue</td>
</tr>
<tr>
<td>60 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0160</td>
<td>For general culture</td>
</tr>
<tr>
<td>100 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0010</td>
<td>For cleaning tissue</td>
</tr>
<tr>
<td>Cryogenic box </td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5026-1010</td>
<td>&nbsp;</td>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5100-0001</td>
<td>"Mr. Frosty"</td>
</tr>
<tr>
<td>2.0 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5012-0020</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>D128-500</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>Ethanol 200 proof</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E7023</td>
<td>For sterilizing razor blade</td>
</tr>		</tbody>
		</table>
		</div>

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

Neste estudo, descrevemos um protocolo padronizado para a criopreservação e descongelamento de blocos de tecido cortical do cérebro para gerar altamente enriquecido culturas neuronal. Para este protocolo o meio de congelamento utilizado é de 10% dimetil sulfóxido (DMSO) diluído em solução de Hank Sal Buffered (HBSS). Blocos de tecido cortical são transferidos para criotubos contendo o meio de congelamento e, lentamente, congelados a -1 ° C / min em um recipiente taxa controlada de congelamento. Pós-descongelamento de processamento e dissociação de blocos de tecido congelado consistentemente produzidos neuronal enriquecido culturas que apresentaram crescimento rápido durante neuríticas os primeiros 5 dias em cultura e expansão significativa da rede neuronal em 10 dias. Coloração imunocitoquímica com o marcador glial fibrilar astrocíticos proteína ácida (GFAP) e do marcador neuronal beta-tubulina classe III, revelou um alto número de neurônios e astrócitos em culturas. Geração de culturas de células precursoras neurais após a dissociação do tecido bloco resultou em rápida expansão neurospheres, que produziu um grande número de neurônios e astrócitos em condições de diferenciação. Este protocolo de criopreservação simples permite a preservação rápido, eficiente e barata de blocos de tecido cortical do cérebro, que concede uma maior flexibilidade para a geração posterior de culturas de células neuronais, astrócitos, e neuronal precursor.

Watch this Scientific Journal Video about Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures at JoVE.com

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Aqui, nós descrevemos um método eficiente para criopreservação e descongelamento de blocos de tecido cortical do cérebro para gerar altamente enriquecido culturas neuronal. Este protocolo simples, fornece flexibilidade para a geração posterior de culturas de células neuronais, astrócitos, e neuronal precursor.

Criopreservação de tecido cortical Blocos de Geração de culturas altamente enriquecido Neuronal

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

cryopreservation-cortical-tissue-blocks-for-generation-highly

Research

JoVE Journal

Neuroscience

13.5K visualizações.  University of California, Irvine. Aqui, nós descrevemos um método eficiente para criopreservação e descongelamento de blocos de tecido cortical do cérebro para gerar altamente enriquecido culturas neuronal. Este protocolo simples, fornece flexibilidade para a geração posterior de culturas de células neuronais, astrócitos, e neuronal precursor.

Vídeo: Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Geração de células-tronco neurais de Discarded tecido fetal humano Cortical

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neurociência

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multieletrodos Recordings Array of Avalanches Neuronal em culturas organotípicas

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Derivação de Enriquecido Culturas oligodendroglial e Neuron / oligodendroglial Myelinating Co-culturas de tecidos pós-natais murino

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

Um sistema totalmente automatizado e altamente versátil para testes de várias funções cognitivas e registro das atividades neuronais em roedores

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Preparação de Neuronal co-culturas com precisão única célula

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

A incubação prolongada de aguda Neuronal Tissue de Eletrofisiologia e cálcio-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Aumento Time-dependente na resposta da rede à estimulação de culturas de pilha neuronais em matrizes do micro-elétrodo

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Geração livre de células neuronais derivadas do sangue

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

Culturas enriquecidas por cone da retina de embriões de frango para estudar rod a interações celulares cone

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Geração robusta e altamente reprodutível de organoides cerebrais corticais para modelar senescência neuronal cerebral in vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...