Development of this protocol was funded by private donations directed to the Parkinson's Institute Brain Donation Program.

O registro de doação do cérebro inclui a documentação, o registando a sua intenção de doar. A permissão da autópsia para a recuperação do tecido é fornecido pelo parente mais próximo, conforme permitido por lei. Pesquisas utilizando amostras de autópsia recolhidos são analisados ​​pelo conselho de revisão institucional (IRB) para garantir o cumprimento Health Insurance Portability e Accountability Act normas (HIPAA). NOTA: As amostras leptomeninges são recolhidos pelo dissector cerebral ou neuropatologista durante uma dissecção cérebro e são armazenados em 50 tubos cônicos mL contendo 25-30 mL meninges meios de crescimento. A amostra é armazenada a 4 ° C até à preparação da amostra. O tratamento deve ser realizado tão rapidamente quanto possível, a viabilidade das células diminui com o tempo de pós-mortem. 1. Set-up antes de iniciar o leptomeninges Dissection NOTA: As etapas 1,1-1,3 devem ser executadas dentro de uma cabine de segurança biológica. <ol><li> Prepare meio de crescimento meninges através da combinação de 375 mL de Dulbecco Modified Eagle Media, 100 mL de soro bovino fetal, 5 ml 10 mM de aminoácidos não essenciais, 5 mL de 200 mM de L-alanil-L-glutamina, 5 mL de piruvato de sódio 100 mM, 5 ml 10000 U / mL de penicilina / estreptomicina e 5 ml de 250 ug / mL de anfotericina B, em 500 mL unidade de filtro. Filtrar e misturar. Use a mídia por até quatro semanas. </li><li> Coloque todos os materiais na cabine de segurança biológica, antes de iniciar a dissecção: pratos de Petri 4x, 2x placas de 6 poços, 2x escalpelos esterilizados, 15-20 lamelas de 15 mm, tampão fosfato salino (PBS), pipetas serológicas, aspirando pipetas. </li><li> Adicionar 1 ml de solução esterilizada por autoclave de gelatina a 0,1% a cada poço de uma placa de 6 poços. Colocou o prato de lado por 30-60 min. Prepare 2x placas de 6 poços. </li></ol> 2. Preparação de leptomeninges Amostra NOTA: As etapas 2,1-2,3 devem ser executadas dentro de uma cabine de segurança biológica. <ol><li> Adicionar aproximadamente 10 mL de PBS paraa 10 cm de placa de cultura de tecido estéril e colocar a amostra no prato meninges. Utilizando uma pinça, lavar cuidadosamente leptomeninges com PBS para remover o sangue. </li><li> Transferir as meninges para uma nova placa de cultura de tecidos de 10 cm contendo 10 ml de PBS fresco e continuar a lavar. Repita como necessário para remover o máximo de sangue possível. </li><li> Usando dois bisturis, cortados leptomeninges tecido em uma cerca de 6 cm x 6 cm grande pedaço. </li><li> Coloque a tampa sobre um prato de cultura e transferência para um microscópio de dissecação numa câmara de fluxo laminar horizontal. </li></ol> 3. Dissecção da leptomeninges Tissue NOTA: As etapas 3,1-3,3 devem ser executadas dentro de uma câmara de fluxo laminar horizontal usando um microscópio de dissecação. <ol><li> Remover os vasos sanguíneos a partir de uma região sobre a peça leptomeninges (passo 2.3) suficientemente grande para criar pelo menos 20-25 3 peças mm x 3 mm. Segure as leptomeninges em lugar com um bisturi e com a outra, usar um mo raspagem suave e superficialção para separar os vasos. </li><li> Cortar pequenos quadrados de tecido da região preparado. Corte em um processo passo a passo pela primeira confecção de peças maiores e, em seguida, cortando os em seções menores. </li><li> Usar um bisturi para manter o tecido no lugar e outra bisturi para cortar o tecido com um movimento de balanço. Devido à consistência do tecido, é difícil para deslizar os bisturis uns contra os outros. Isso só vai rasgar o tecido distante e resulta em bordas irregulares. </li><li> Continue cortando as peças pela metade até que haja cerca de 20-25 peças 3 mm x 3 mm. </li></ol> 4. Transmissão de dissecado meninges partes em placas de cultura de tecidos NOTA: Passo 4 deve ser efectuado dentro de uma cabine de segurança biológica. <ol><li> gelatina aspirado a partir da placa de 6 poços (a partir do passo 1.3). Adicionar 1 mL de meio de crescimento meninges a cada poço. Utilizando uma pinça estéril, colocar 3-4 peças de biópsia em cada poço. </li></ol> 5. A colocação de tampa desliza sobre Meninges Pieces NOTA: As etapas 5,1-5,2 devem ser executadas dentro de uma câmara de fluxo laminar horizontal usando um microscópio de dissecação. <ol><li> Cubra os pedaços com 1-2 lamelas circulares de 15 mm estéreis por poço. </li><li> Pressionar firmemente com uma pinça para garantir que as peças são tocar no fundo da placa. Isto permite a fixação das peças de tecido para o fundo do poço. </li><li> Colocar a placa de 6 poços a uma temperatura de 37 ° C, 5% de CO2. </li></ol> Manutenção Cultura 6. celular NOTA: As etapas 6,1-6,3 devem ser executadas dentro de uma cabine de segurança biológica. Lidar com placas de cultura com cuidado como lamelas não deve se mover e deslocar as peças meninges. <ol><li> Após 2 dias em cultura, adicionar um adicional de 1 ml de meio de crescimento fresco as meninges a cada poço. </li><li> No dia 7, aspirar cuidadosamente os meios de comunicação e adicionar 2 mL de meio fresco a cada poço. </li><li> Após o Dia 9, continuam a mudar meios de crescimento a cadaoutro dia até que a cultura se torna confluente. </li></ol> 7. Passaging NOTA: Todas as etapas devem ser executadas dentro de uma cabine de segurança biológica. Uma vez que os fibroblastos meníngeos ter migrado para fora dos pedaços de tecido das meninges e começam a crescer em direcção as bordas do recipiente de cultura, expandir fibroblastos meníngeos em maior placa de cultura. <ol><li> Antes de iniciar o Passaging, revestir a placa de cultura com 1% de gelatina durante 15-30 min. </li><li> media aspirado de 6 poços prato e lavar as células com 2 ml PBS. </li><li> Adicionar 1 ml de tripsina / EDTA a cada poço e incubar a 37 ° C durante 5-10 min. Não é necessário retirar as peças de cobertura ou do tecido meníngeo para este passo. </li><li> Quando as células são arredondadas e começar a destacar, adicionar 2 mL de meio de cultura. </li><li> células de pipeta cima e para baixo para interromper completamente todos os aglomerados e transferir a solução de célula para maior prato de cultura. </li><li> Usar uma proporção de 1: 3 a 1: 4 para combinar as células a partir de três pratos de 6 poços empara uma placa de cultura T75. </li><li> Uma vez que as células de fibroblastos meníngeas estão confluentes em frascos de cultura T75, trypsinize as células, e criopreservar em meios de criopreservação a 1x10 6 células / ml por frasco. Um balão de cultura T75 com fibroblastos meninges confluentes contém cerca de 5-7 milhões de células. </li></ol> 8. Caracterização dos fibroblastos Meníngeas por imunocoloração NOTA: Antes de iniciar o procedimento, preparar solução de paraformaldeído a 4%, soro de cabra normal a 5% em PBS (tampão de bloqueio), 0,3% de Triton X-100 em PBS (tampão de permeabilização), soluções de anticorpos primários e secundários, e 1 ug / mL de Hoechst 33342 diluído em PBS a partir de 10 mg estoque / mL. <ol><li> Semente de 15.000 a 30.000 fibroblastos meníngeos em slides câmara de 8 poços revestidos com gelatina a 0,1%. </li><li> Depois de 24 h, quando as células são ligadas, fixar as células em 100 uL de paraformaldeído a 4% por poço, durante 10 min à temperatura ambiente. Lavar os poços 3 vezes com PBS 1x. </li><li> Permeabilizar as células com 150 uL de 0,3% de Triton X-100 durante 5 min à temperatura ambiente. Lavar os poços 3 vezes com PBS 1x. </li><li> Adicionar 200 uL de solução de bloqueio (PBS + 5% de soro normal de cabra) durante 1 h à temperatura ambiente. </li><li> Aspirar a solução de bloqueio e adicionar 150 uL de anticorpos primárias desejadas diluído em solução de bloqueio. Para double-coloração, prepare diluições de trabalho dos anticorpos primários em tubos de centrífuga separados no gelo como se segue: <ol><li> Prepare a diluição de 1: 300 de anti-fibronectina + 1: 200 de diluição de anti-nestina em solução de bloqueio. Prepare diluição 1: 250 de anti-SERPINH1 + 1: 1000 diluição de anti-TUJ1 em solução de bloqueio. Prepare 1: 250 de diluição de anti-SERPINH1 + 1: 100 de diluição de anti-SOX2 em solução de bloqueio </li></ol></li><li> Incubar a 4 ° C durante a noite. </li><li> Lavar os poços 3 vezes com PBS 1x. </li><li> Prepare um trabalho diluição 1: 400 de fluorescente etiquetado anticorpos secundários de cabra anti-coelho (verde; Ex / Em 2 </saté&gt; 495/519 nm) e de cabra anti-ratinho (vermelho; Ex / Em 590/617 nm 2) em solução de bloqueio e adicionar 100 uL a cada poço. Incubar durante 1 h, no escuro, à temperatura ambiente. Em seguida, lave os poços uma vez com PBS 1x. </li><li> Adicionar 100 uL de 1 ug / ml de Hoechst 33342 (azul; Ex / Em 358/461 nm 2) solução a cada poço e incuba-se no escuro durante 3 min. Lavar os poços 3 vezes com 1x PBS, deixando na final de lavagem 1x PBS no poço, e cobrir o slide com folha de alumínio. </li><li> Analise as células sob microscópio fluorescente. </li></ol>

<ol>
	<li>Decimo, I., Fumagalli, G., Berton, V., Krampera, M., Bifari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meninges:+from+protective+membrane+to+stem+cell+niche.">Meninges: from protective membrane to stem cell niche.</a> Am J Stem Cells. 1 (2), 92-105 (2012).</li><li>Bifari, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+stem/progenitor+cells+with+neuronal+differentiation+potential+reside+in+the+leptomeningeal+niche.">Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche.</a> J Cell Mol Med. 13 (9B), 3195-3208 (2009).</li><li>Bifari, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meninges+harbor+cells+expressing+neural+precursor+markers+during+development+and+adulthood.">Meninges harbor cells expressing neural precursor markers during development and adulthood.</a> Front Cell Neurosci. 9, 383 (2015).</li><li>Decimo, I., Bifari, F., Krampera, M., Fumagalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+niches+in+health+and+diseases.">Neural stem cell niches in health and diseases.</a> Curr Pharm Des. 18 (13), 1755-1783 (2012).</li><li>Hayashi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meningeal+cells+induce+dopaminergic+neurons+from+embryonic+stem+cells.">Meningeal cells induce dopaminergic neurons from embryonic stem cells.</a> Eur J Neurosci. 27 (2), 261-268 (2008).</li><li>Nakagomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptomeningeal-derived+doublecortin-expressing+cells+in+poststroke+brain.">Leptomeningeal-derived doublecortin-expressing cells in poststroke brain.</a> Stem Cells Dev. 21 (13), 2350-2354 (2012).</li><li>Hjelm, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+autopsy+donor-derived+somatic+cells.">Induction of pluripotent stem cells from autopsy donor-derived somatic cells.</a> Neurosci Lett. 502 (3), 219-224 (2011).</li><li>Hjelm, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-differentiated+neural+cell+cultures+progress+towards+donor-identical+brain+tissue.">In vitro-differentiated neural cell cultures progress towards donor-identical brain tissue.</a> Hum Mol Genet. 22 (17), 3534-3546 (2013).</li><li>Meske, V., Albert, F., Wehser, R., Ohm, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+autopsy-derived+fibroblasts+as+a+tool+to+study+systemic+alterations+in+human+neurodegenerative+disorders+such+as+Alzheimer's+disease--methodological+investigations.">Culture of autopsy-derived fibroblasts as a tool to study systemic alterations in human neurodegenerative disorders such as Alzheimer's disease--methodological investigations.</a> J Neural Transm (Vienna). 106 (5-6), 537-548 (1999).</li><li>Bliss, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+postmortem+human+dura+mater+and+scalp+for+deriving+human+fibroblast+cultures.">Use of postmortem human dura mater and scalp for deriving human fibroblast cultures.</a> PLoS One. 7 (9), e45282 (2012).</li><li>Sproul, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+iPSC+lines+from+archived+non-cryoprotected+biobanked+dura+mater.">Generation of iPSC lines from archived non-cryoprotected biobanked dura mater.</a> Acta Neuropathol Commun. 2, 4 (2014).</li><li>Adler, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+clinical+diagnostic+accuracy+of+early+vs+advanced+Parkinson+disease:+clinicopathologic+study.">Low clinical diagnostic accuracy of early vs advanced Parkinson disease: clinicopathologic study.</a> Neurology. 83 (5), 406-412 (2014).</li><li>Hughes, A. J., Daniel, S. E., Kilford, L., Lees, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+clinical+diagnosis+of+idiopathic+Parkinson's+disease:+a+clinico-pathological+study+of+100+cases.">Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases.</a> J Neurol Neurosurg Psychiatry. 55 (3), 181-184 (1992).</li><li>Hughes, A. J., Daniel, S. E., Lees, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+accuracy+of+clinical+diagnosis+of+Lewy+body+Parkinson's+disease.">Improved accuracy of clinical diagnosis of Lewy body Parkinson's disease.</a> Neurology. 57 (8), 1497-1499 (2001).</li><li>Langston, J. W., Sch&#252;le, B., Rees, L., Nichols, R. J., Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisystem+Lewy+body+disease+and+the+other+parkinsonian+disorders.">Multisystem Lewy body disease and the other parkinsonian disorders.</a> Nature Genetics. 47 (12), 1378-1384 (2015).</li><li>Vangipuram, M., Ting, D., Kim, S., Diaz, R., Sch&#252;le, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+punch+biopsy+explant+culture+for+derivation+of+primary+human+fibroblasts.">Skin punch biopsy explant culture for derivation of primary human fibroblasts.</a> J Vis Exp. (77), e3779 (2013).</li><li>Byers, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNCA+triplication+Parkinson's+patient's+iPSC-derived+DA+neurons+accumulate+alpha-synuclein+and+are+susceptible+to+oxidative+stress.">SNCA triplication Parkinson's patient's iPSC-derived DA neurons accumulate alpha-synuclein and are susceptible to oxidative stress.</a> PLoS One. 6 (11), e26159 (2011).</li><li>Sanders, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+mutations+cause+mitochondrial+DNA+damage+in+iPSC-derived+neural+cells+from+Parkinson's+disease+patients:+reversal+by+gene+correction.">LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.</a> Neurobiol Dis. 62, 381-386 (2014).</li><li>Beevers, J. E., Caffrey, T. M., Wade-Martins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+(iPSC)-derived+dopaminergic+models+of+Parkinson's+disease.">Induced pluripotent stem cell (iPSC)-derived dopaminergic models of Parkinson's disease.</a> Biochem Soc Trans. 41 (6), 1503-1508 (2013).</li></ol>

The authors have nothing to disclose.

Este protocolo descreve um protocolo simples e robusto para derivar de uma cultura de fibroblastos a partir de meninges leptomeninges postmortem humanos recolhidos em conjunto com uma doação cérebro. Há muito poucas descrições de protocolos para derivar culturas de células a partir de material humano post-mortem. Dois estudos descrevem culturas de fibroblastos 7, 8, derivados de pele 9, um estudo descreve amostras da dura 10, e outro descreve amostras da dura congelada não criopreservadas 11. Nós preparar duas placas de 6 poços com 2-3 pedaços de tecido (cerca de 3 mm x 3 mm) em cada poço para assegurar crescimento e expansão suficiente antes da criopreservação. É fundamental que as peças meninges estão ligados à placa de cultura por gentil pressionando uma lamela de vidro estéril sobre os pedaços de tecido para segurá-los no lugar. Sem uma lamela, as meninges peças não seráT Encaixe para o fundo da placa de cultura. excrescência sucesso apenas é observada quando os pedaços de tecido está em contacto directo com a parte inferior de plástico. Se as peças de tecido flutuar no meio de cultura, sem crescimento celular a partir do tecido é observada. Este deve ser realizado com cerca de 500 ul de meios de crescimento de não secar as amostras e, em seguida, adicionar-se a 1 mL de meio de crescimento para o recipiente. Não é necessário o uso de mais de silicone para segurar as peças de cobertura no lugar. Se as peças são menores do que 3 mm x 3 mm, o crescimento é mais lenta e, em alguns casos, pode ser estabelecida não há culturas confluentes. É crítico para crescer as células não muito escasso e recomenda-se uma razão de divisão de 1: 3 a 1: 4. pequenos poços individuais permitir uma melhor gestão possível contaminação. Wells podem ser alimentados individualmente e pipetas devem ser alterados entre as cavidades. Temos derivada com sucesso fibroblastos meníngea de 9 amostras post-mortem com um postmortem intervalo entre 10-24 h. Os doadores tinham entre 70 e 88 anos de idade e ter durações doença da doença de Parkinson entre os 8-31 anos. Notamos que os tecidos com um intervalo mais longo post-mortem demorar mais tempo antes que as primeiras células crescem para fora (até 9 dias). Em amostras com mais curto intervalo post mortem, observa-se crescimento logo 3 dias após a preparação. Nós não ter notado uma diferença no crescimento com base na idade do doador, mas só tivemos doadores&gt; 70 anos. O significado do método é a derivação de uma cultura de células primária para uma doença neurodegenerativa esporádica como a doença de Parkinson, o qual só pode actualmente ser confirmada na autópsia 12, 13, 14, uma vez que não está definindo não totalmente características clínicas, técnicas de imagiologia ou biomarcadores que possam fornecer um diagnóstico definitivo durante a vida 15. Temos anteriormente desenvolvido um protocolo para a derivação de fibroblastos de biópsias de pele, principalmente para casos geneticamente confirmado de doença de 16 de Parkinson, que nos permitiram gerar células-tronco pluripotentes induzidas e estudar mecanismos da doença 17, 18, mas este novo protocolo será crucial para comparar diretamente alterações neuropatológicas com os resultados in vitro para a doença esporádica de Parkinson. Para o nosso conhecimento, este é o primeiro protocolo decorrente fibroblastos meninges de leptomeninges humanos post-mortem. Culturas de fibroblastos Meníngeas pode ser directamente utilizada para estudar os mecanismos moleculares relativas à doença ou lesão ou como uma fonte de reprogramação nuclear em células estaminais pluripotentes induzidas ou a conversão directa em culturas neuronais como modelos pré-clínicos avançados de doença esporádica de Parkinson ou outras doenças neurodegenerativas, 19. estes humodelos derivados do paciente homem fornecem a base e acelerar avanços na medicina personalizada e regenerativa.

Meninges constituído por três membranas que protegem o cérebro: dura-máter máter, aracnóide e pia. Mais recentemente, tem sido reconhecido que as meninges também desempenham um papel importante no desenvolvimento do cérebro e do cérebro 1 homeostase. Meninges são derivadas de mesênquima e células derivadas da crista neural e interessantemente, foi mostrado que as células progenitoras que residem em meninges pode dar origem a neurónios in vitro e in vivo após o transplante 2, 3, 4. Culturas meninges foram também utilizados com sucesso como camadas alimentadoras, como eles possuem actividade indutora derivada de células de estroma para a diferenciação de células estaminais embrionárias em neurónios dopaminérgicos 5. Além disso, leptomeninges têm o potencial para se diferenciar em neurónios directamente, astrócitos, oligodendrócitos e sob condições isquémicas 6. Para este protocolo, as amostras de meninges pós-morte humanos são recolhidos a partir do aracnóide e pia, chamados coletivamente os leptomeninges, e são adquiridos como parte de uma doação cérebro humano para fins de pesquisa. Dissecção do cérebro é realizada dentro de 24 h da morte e da amostra leptomeninges é colocado em meio de cultura frio para posterior processamento dentro do próximo 6-8 h, conforme ilustrado aqui neste protocolo. Este protocolo descreve a dissecção e a preparação de amostras de meninges humanos para o desenvolvimento de culturas de células primárias leptomeninges paciente. O tecido é cortado em pedaços de aproximadamente 25-30 3 mm x 3 mm quadrados. Três peças são colocadas em cada poço de 6 poços e realizou-se com lamelas de vidro redondas revestidas com gelatina. A dissecção meninges leva cerca de 25-35 minutos. O principal desafio com esta cultura é a esterilidade como a aquisição cérebro, transporte e dissecção geralmente não são realizados em condições estéreis. Tor conseguinte, é importante para completar o meio de cultura com um cocktail de penicilina, estreptomicina, e anfotericina B e usar pratos de poços múltiplos para separadamente pedaços de tecido de cultura. Crescimento de fibroblastos meníngeos ocorre geralmente dentro da primeira semana. Meios é mudada a cada dois ou três dias até que as células estão confluentes, e as células são passadas enzimaticamente. Os fibroblastos meninges são criopreservadas em 1 milhão de células por mL / recipiente em mídia criopreservação. Com este protocolo, 20-30 milhões de fibroblastos meninges pode ser derivado de 6-8 semanas para a criopreservação. aplicações a jusante desses fibroblastos meninges são culturas primárias para a pesquisa da doença, a diferenciação neuronal direta ou derivação de células-tronco pluripotentes induzidas a partir das leptomeninges para a compreensão dos mecanismos da doença e para o desenvolvimento de drogas.

<table border="0" cellpadding="0" cellspacing="0" width="1256">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:459px;">Corning Petri dishes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:127px;">351029</td>
			<td style="width:485px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc 6-well plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-mm cover slips</td>
			<td>Fisher Scientific</td>
			<td colspan="2">12-545-83 15CIR-1D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels, sterile blade, No. 15</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved precision tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>22-230490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution</td>
			<td>Fisher Scientific</td>
			<td>SH30264.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 500 mL filter unit</td>
			<td>Fisher Scientific</td>
			<td>430770</td>
			<td>Combine media components and filter.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm2&#160;</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Growth Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hyclone DMEM</td>
			<td>Fisher Scientific</td>
			<td>SH30081.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone FBS</td>
			<td>Fisher Scientific</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B (Yellow Solution/250 &#181;g/mL)</td>
			<td>Fisher Scientific</td>
			<td>BP264520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bambanker Freeze 120 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9582225</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fibronectin Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">8 well chamber slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>1256518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Glycerol&#160;</td>
			<td>BioRad</td>
			<td>9455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% normal goat serum&#160;</td>
			<td>Fisher Scientific</td>
			<td>101098-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Fibronectin antibody [F1]</td>
			<td>Abcam</td>
			<td>ab32419</td>
			<td>1:300 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SERPINH1</td>
			<td>Sigma</td>
			<td>S5950-200ul</td>
			<td>1:250 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SOX2</td>
			<td>Millipore</td>
			<td>MAB4343</td>
			<td>1:100 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td>1:200 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TUJ1</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>1:1000 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 anti-rabbit</td>
			<td>Thermo Fisher</td>
			<td>A11029</td>
			<td>1:400 dilution in blocking solution; (green channel; Ex/Em2 495/519 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 555 anti-mouse</td>
			<td>Thermo Fisher</td>
			<td>A21424</td>
			<td>1:400 dilution in blocking solution; (red channel; Ex/Em2 590/617 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td>dilute to a final concentration of 1.0 &#956;g/ml; (blue channel; Ex/Em2 358/461 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Suppliers are suggestions, similar products from alternative vendors can be used as well.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation of leptomeninges explant cultures from postmortem human brain donors

Apesar de grandes progressos foram feitos na caracterização clínico da doença de Parkinson, vários estudos relatam que o diagnóstico da doença de Parkinson não é patologicamente confirmada em até 25% da doença clinicamente diagnosticada de Parkinson. Portanto, o tecido coletadas de pacientes com diagnóstico clínico de doença de Parkinson idiopática pode ter uma alta taxa de erros de diagnóstico; portanto, os estudos in vitro a partir de tais tecidos para estudar a doença de Parkinson como um modelo pré-clínico pode tornar-se inútil. Através da recolha leptomeninges humanos pós-mortem com um diagnóstico neuropatológico confirmado de doença de Parkinson e caracteriza-se por perda de células nigroestriatal e inclusões de proteínas intracelulares denominadas corpos de Lewy, pode-se ter a certeza de que o parkinsonismo clinicamente observada não é causado por um outro processo de doença subjacente (por exemplo, de tumores, arteriosclerose). Este protocolo presents a dissecação e preparação de leptomeninges humanos pós-morte para derivação de uma cultura de fibroblastos meníngea. Este processo é robusto e tem uma elevada taxa de sucesso. O desafio da cultura é esterilidade como a aquisição cérebro geralmente não é realizado sob condições estéreis. Portanto, é importante para completar o meio de cultura com um cocktail de penicilina, estreptomicina, e anfotericina B. A derivação de fibroblastos meninges de casos de autópsia-confirmadas com a doença de Parkinson fornece a base para a modelagem in vitro da doença de Parkinson. fibroblastos meninges aparecem 3-9 dias após a preparação da amostra e cerca de 20-30 milhões de células podem ser criopreservados em 6-8 semanas. A cultura de fibroblastos meníngea é homogéneo e as células expressam fibronectina, um marcador comumente usado para identificar as meninges.

Quando o protocolo de processamento tem sido bem sucedida leptomeninges, crescimento de fibroblastos meníngeos primeiro se observa três a nove dias após a dissecação, embora isso possa depender do comprimento do intervalo pós-mortem de cérebro. A Figura 1 demonstra culturas de fibroblastos meninges de quatro doadores diferentes. A Figura 1A mostra um pedaço leptomeninges pressionada por uma lamela de vidro (linha diagonal escuro) e crescimento de fibroblastos em torno dos tecidos quatro dias após o processamento de um doador de 88 anos de idade, com 10 h 20 min de intervalo post-mortem. A Figura 1B mostra esparso fibroblastos excrescência sete dias pós-processamento de um doador de 70 anos de idade, e 11 h 45 min intervalo post mortem. A Figura 1C mostra fibroblastos excrescência 13 dias após dissecção a partir de um doador de 88 anos de idade, e 24 h de intervalo post mortem. A Figura 1D mostra uma cultura confluente foi passada em que uma vess T75EL e as células podem ser criopreservadas nesta fase. Fibroblastos meninges são bipolar ou multipolar e ter alongado ou formas irregulares (Figura 2). Eles são caracterizados pela expressão de glicoproteína de fibronectina e marcador de fibroblastos SERPINH que está localizada no retículo endoplasmático (Figuras 2A-2C). Fibroblastos Meníngeas não são imuno-reactivos com o factor de transcrição SRY (Sexo Região Determinante Y) -Box 2 (SOX2) que é um marcador para as células-tronco indiferenciadas (Figura 2A). No entanto, um subconjunto de fibroblastos leptomeníngeas é positiva para o tipo VI nestina filamento intermediário, um marcador de células estaminais neuronais (Figura 2B), e classe III específica dos neurónios beta-tubulina (TUJ1) (Figura 2C). <img alt="figura 1" src="/files/ftp_upload/55045/55045fig1.jpg" /> Figura 1.Exemplos de crescimento de fibroblastos meníngeos de quatro doadores diferentes. A) pedaço de tecido leptomeninges é pressionada por uma lamela de vidro (linha diagonal escuro) e crescimento de fibroblastos ao redor do tecido foi observado quatro dias após o processamento de um doador de 88 anos de idade, com 10 h 20 min de intervalo post-mortem. B) fibroblastos Dispersa conseqüência sete dias pós-processamento de um doador de 70 anos de idade e 11 h 45 min de intervalo post-mortem. C) crescimento de fibroblastos meníngea 13 dias após a dissecação de um doador de 88 anos de idade, e 24 h de intervalo post mortem. D) confluentes cultura de fibroblastos meníngea passadas para um vaso de T75. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55045/55045fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/55045/55045fig2.jpg" /> Figura 2. Immunost<html> aining para meninges, células-tronco, células-tronco neurais, e marcadores de neurônios. A) imunofluorescência para os marcadores de fibroblastos SERPINH1 (verde) e haste marcador de células SOX2 (vermelho). SERPINH1 foi expressa em todos os núcleos celulares contados. Caule SOX2 marcador de células não foi detectada em cultura estas leptomeninges. B) imunofluorescência para fibronectina-meninges específicas marcador (verde) e neural marcador de células-tronco nestina (vermelho). 52,1% de núcleos de células contados eram positivos tanto para nestina e fibronectina. C) imunofluorescência para SERPINH1 (verde) e TUJ1 marcador neuronal (vermelho). 6,9% dos núcleos contados foram positivos para SERPINH1 e TUJ1. dados de contagem de células foram determinadas usando o manual celular Contra Multi-Select ferramenta em ImageJ. imagens coradas individuais foram avaliadas através da contagem do número de células DAPI-positiva em comparação com o número de células positivas para cada marcador, em percentagens.lank &quot;&gt; Por favor clique aqui para ver uma versão maior desta figura. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Dia </td><td colspan="6"> Resultados esperados </td></tr><tr><td> 3-9 </td><td colspan="6"> Conseqüência dos primeiros fibroblastos meninges. </td></tr><tr><td> 7-18 </td><td colspan="6"> fibroblastos meninges expandir, e a cultura se torna mais denso. Troca de mídia de 2 ml a cada dois dias. </td></tr><tr><td> 18-25 </td><td colspan="6"> placa de 6 poços se torna confluente, uma vez fibroblastos meníngeas estão confluentes combinar três poços em um balão de cultura T75 (passagem de 1: 3 a 1: 4). </td></tr><tr><td> 26-45 </td><td colspan="6"> Um balão T75 confluente cultura resulta em 5-7 milhões de células. Criopreservação de fibroblastos meninges em 1 milhão de células / frasco. </td></tr></tbody></table> Abale 1: Cronologia da excrescência celular.

Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

O protocolo de cultura leptomeninges explante a partir de cérebro pós-morte humana é uma forma tecnicamente robusta e simples para derivar fibroblastos meninges fibronectina-positiva dentro de 6-8 semanas e criopreservação de aproximadamente 20-30 milhões de células.

Derivação de culturas leptomeninges explantes de Postmortem Cérebro humano Doadores

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

derivation-leptomeninges-explant-cultures-from-postmortem-human-brain

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.2K visualizações.  Parkinson’s Institute and Clinical Center.  O protocolo de cultura leptomeninges explante a partir de cérebro pós-morte humana é uma forma tecnicamente robusta e simples para derivar fibroblastos meninges fibronectina-positiva dentro de 6-8 semanas e criopreservação de aproximadamente 20-30 milhões de células. 

Vídeo: Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

Labeling DiOLISTIC de Neurônios de roedores e não-humanos fatias do cérebro do primata

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

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 ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivação eficiente dos recursos humanos Progenitores Neuronal e Neurônios de pluripotentes células estaminais embrionárias humanas com a indução de moléculas pequenas

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

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 ...

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

Derivação da glia Precursores restritos de camundongos E13

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Neurais culturas de explante de<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Progenitor derivado Sistema de Cultura oligodendroglial Cérebro Fetal Humano

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivação sem alimentador de células da crista neural progenitoras pluripotentes humanas de células-tronco

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

De alta resolução de ressonância magnética estrutural do Subcórtex Humano<I&gt; In Vivo</I&gt; E Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Isolamento dos capilares cerebrais do tecido do cérebro humano fresco

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...