Name: culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research
Uploaded: 2016-06-10T13:00:00.000+00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Os autores gostariam de agradecer a equipe Biospherix por sua ajuda em aprender a usar o sistema de cultura celular Xvivo fechado, especialmente Matt Freeman; o pessoal do Miles &amp; Kelley Construction Company, Inc. para o seu trabalho na criação da infra-estrutura laboratorial, especialmente Russ Hughes; o pessoal do Hospital Infantil de departamento de Orange County de Instalações e Serviços de Suporte para o seu trabalho na coordenação da remodelação de laboratório, especialmente Adam Lukhard e Devin Hugie; o pessoal do Hospital Infantil de departamento de Orange County de Sistemas de Informação pela sua ajuda na criação da infra-estrutura de gerenciamento de dados e acesso remoto, especialmente Viet Tran; Hospital Infantil de Orange County Equipe de Gestão Executiva por seu apoio de longa data do projeto, especialmente Dr. Maria Minon e Brent Dethlefs. Este trabalho foi financiado pelo Hospital Infantil de Orange County e do California Institute for Regenerative Medicine através TR3-05476 subvenção para PHS. Todos os autores contribuíram igualmente para este trabalho.

<ol>
	<li>Brick, D. J., 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=The+Autism+Spectrum+Disorders+Stem+Cell+Resource+at+Children's+Hospital+of+Orange+County:+Implications+for+Disease+Modeling+and+Drug+Discovery.">The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.</a> Stem Cells Transl.Med. 3 (11), 1275-1286 (2014).</li><li>Nethercott, H. E., Brick, D. J., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+induced+pluripotent+stem+cells+by+lentiviral+transduction.">Derivation of induced pluripotent stem cells by lentiviral transduction.</a> Methods Mol.Biol. 767, 67-85 (2011).</li><li>Pistollato, 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=Hypoxia+and+HIF1alpha+repress+the+differentiative+effects+of+BMPs+in+high-grade+glioma.">Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.</a> Stem Cells. 27 (1), 7-17 (2009).</li><li>Pistollato, F., Chen, H. L., Schwartz, P. H., Basso, G., Panchision, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+controls+the+expansion+of+human+CNS+precursors+and+the+generation+of+astrocytes+and+oligodendrocytes.">Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes.</a> Mol.Cell Neurosci. 35, 424-435 (2007).</li><li>Raval, J. S., Koch, E., Donnenberg, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+non-viable+airborne+particles+correlates+with+airborne+colonies+and+represents+an+acceptable+surrogate+for+daily+assessment+of+cell-processing+cleanroom+performance.">Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance.</a> Cytotherapy. 14 (9), 1144-1150 (2012).</li><li>Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+stem+cell+therapies+for+neurological+diseases.">The potential of stem cell therapies for neurological diseases.</a> Expert.Rev.Neurother. 6, 153-161 (2006).</li><li>Schwartz, P. H., Brick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapies+for+the+lysosomal+storage+diseases+-+the+quintessential+neurodegenerative+diseases.">Stem cell therapies for the lysosomal storage diseases - the quintessential neurodegenerative diseases.</a> Curr.Stem Cell Res.Ther. 3 (2), 88-98 (2008).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol.Biol. 767, 107-123 (2011).</li><li>Stover, A. 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=Process-based+expansion+and+neural+differentiation+of+human+pluripotent+stem+cells+for+transplantation+and+disease+modeling.">Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.</a> J Neurosci.Res. 91 (10), 1247-1262 (2013).</li><li>Stover, A. E., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging.">Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.</a> Methods Mol.Biol. 767, 137-146 (2011).</li><li>Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5 (3), 237-241 (2009).</li></ol>

The authors declare that they have no competing financial interests.

As células cultivadas dentro das CPF não vejo mudanças nas concentrações de oxigênio ou dióxido de carbono que se deslocam de incubadora para o processamento de câmara para microscópio de câmara e volta. É crítico que as condições em cada uma das câmaras são combinadas para a incubadora particular em que as células são mantidas antes que as células são removidas da incubadora. A atmosfera dentro do aparelho é continuamente HEPA-filtrado e é personalizável no que diz respeito às concentrações de oxigênio e dióxido de carbono. As células podem ser cultivadas em concentrações normais para unidades ou NSC, 5% e 9%, respectivamente; ou concentrações alternativos podem ser escolhidos para diferentes tipos de células ou para experiências específicas. Assim, o aparelho é fornecido com fontes de oxigénio constante de grau médico, dióxido de carbono e azoto (Figura 4). Todos os três destes gases são fornecidos por sistemas de colector de gás específico do que assegurar o abastecimento constante. O aparelho é também fornecido com uma mistura de gás de calibração consiste em10% (± 0,01%) de dióxido de carbono em oxigénio. Os sistemas de manifold estão alojados fora da unidade de produção de células e os gases são canalizados para a instalação através do teto. O gás de calibração está alojado no interior da instalação. O aparelho é fornecido adicionalmente com aspirador de casa, também através do teto. Usando um sistema de monitoramento eletrônico e sem fio unidades envio, as pressões de todos os colectores de saída são constantemente monitorados. No caso em que qualquer pressão cai fora do intervalo, os operadores das instalações de produção de células são automaticamente telefonou e notificada de tal forma que medidas apropriadas possam ser tomadas. Os requisitos de energia do aparelho são atendidas por seis dedicados 120 V circuitos que descem do teto e ligados a geradores de back-up do hospital para garantir um fornecimento constante. O funcionamento do aparelho é controlado via software em um computador baseado em PC alimentado através de uma fonte de alimentação ininterrupta. Estes arranjos de poder e de computadorassegurar que o sistema funciona continuamente, mesmo em caso de falha do sistema do poder público. O software que controla o aparelho tem uma interface gráfica amigável (Figura 1) que permite o controlo das concentrações de oxigênio e dióxido de carbono, bem como pressões de temperatura, umidade, e de câmara. Os valores de todos estes parâmetros são registadas continuamente para fornecer um registro de execução de todos os parâmetros do aparelho. Este dados são copiados para um servidor remoto a cada noite para proteger a sua integridade. O computador eo software pode ser acessado remotamente por usuários administrativos para avaliar e / ou alterar qualquer parâmetro. Além disso, o computador eo software pode ser acessado remotamente, permitindo avaliação interativa de parâmetros de aparelhos e solução de problemas com os usuários locais. Uma unidade de envio de alarme adicional é ligado ao aparelho de tal modo que os operadores das instalações de produção de células são notificados de qualquer condição fora-de-gama do aparelho. O acesso remoto capabilities permitir o login e avaliação das especificidades da condição fora-de-gama. O aparelho é concebido como um sistema modular tanto em uma macro e micro um sentido. módulos de cultura de células individuais, tais como incubadoras e câmaras de processamento, podem ser personalizadas no que diz respeito às suas dimensões e os requisitos, bem como na sua disposição com respeito um ao outro. Além disso, a maior parte das funções de controlo dos módulos individuais são eles próprios modular de tal modo que os controladores de gás atmosféricos, por exemplo, pode ser facilmente substituído sem perturbação significativa para o sistema. câmaras de processamento especializado, tal como um para visualização microscópica e manipulação de culturas de células, podem ser facilmente adaptado ao sistema. Ambos de contraste de fase e microscópio de fluorescência estão dentro do sistema (Figura 6), de modo que as células podem ser coradas vivo, e as colónias podem ser dissecados nas mesmas condições atmosféricas dentro como tele incubadoras. Encaminhamento de cabos por meio de ilhós seladas nas paredes laterais da câmara de processamento permite que o equipamento, tal como fontes de alimentação e computadores para ser mantido no exterior do aparelho, geralmente sobre um carrinho (Figura 6). As câmaras de processamento na unidade de produção de células têm um padrão de fluxo de ar diferente do BSC convencionais. Em BSCs convencionais, o fluxo de ar flui para baixo a partir de um orifício de evacuação central e se divide em duas correntes separadas, que são então levados até por duas aberturas de entrada diferentes na parte da frente e para trás do piso do armário. Em contraste, a PCF tem uma única abertura na parte da frente do tecto. O ar flui para baixo e em direcção à parte de trás da câmara, onde é, em seguida, conduzido para cima, para uma entrada de ventilação. Embora o CPF é inerentemente muito limpos, este padrão de fluxo de ar única significa que os técnicos têm de ajustar ligeiramente a sua técnica para reduzir o risco de contaminação. Tal como acontece com um BSC convencional, um trabalhador s laboratórioevitar hould colocando suas mãos a montante de placas de cultura de células abertas e garrafas de mídia. No entanto, o sentido a montante é que tenha sido modificado no CPF O laboratório instalação de produção de células em si é bastante padrão e vem equipado com um C congelador -20 °, a -80 ° C freezer, a 4 ° C geladeira, uma centrífuga, e um banho de água. O laboratório também tem uma pia com pedais para operação conveniente mãos livres. Para que este laboratório para se tornar uma unidade de produção de células clínica funcional, no entanto, devem ainda ser feitas várias modificações adicionais. Em primeiro lugar, o aparelho em si tem de ser actualizado para ter a capacidade de monitorar compostos voláteis orgânicos, partículas, e das concentrações de dióxido de cloro que é utilizado para descontaminação. Em segundo lugar, uma câmara de processamento que contém uma máquina de FACS podem ser alojados e ligado ao resto do aparelho através de um módulo de memória intermédia. Isto irá permitir a separação de células e purificação de trpopulações de células ansplantable nas condições ambientais adequadas. Por fim, todo o aparelho deve ser instalado dentro de uma parede macia sala limpa. Isso fornece uma International Organization for Standardization (ISO) Classe 8 ambiente para o aparelho 5. A alta esterilidade e natureza controlado por computador do CPF faz com que seja um sistema ideal para aplicações futuras com a terapia à base de células e os processos de fabrico boas. O risco de contaminação é bastante atenuados, mas o mais importante, as condições de expansão celular são registados automaticamente e arquivados pelo sistema de computador. Desvios em concentrações de gases, temperatura, umidade e todos os eventos de acesso ao sistema são rigorosamente documentadas. Isso pode ajudar muito na investigação de problemas de qualidade do produto. No entanto, ainda há limitações. O uso de qualquer e todos os reagentes e materiais (por exemplo, componentes de mídia, pipetas, placas) deve ser documentada separadamente. Adicionaritionally, há uma infinidade de problemas potenciais (incluindo muitas formas de erro humano) que podem surgir que são completamente alheios às variáveis ​​documentados pelo sistema de monitoramento do CPF. Assim, a necessidade de pessoal altamente treinado e documentação manual detalhado de tarefas permanece no lugar.

Standard stem cell culture techniques suffer from several environmental constraints that place undue stresses on the cells and expose the cells to unacceptable risks of contamination. Among the stresses that cells may endure under standard cell culture conditions are precipitous changes in the levels of carbon dioxide and oxygen concentrations3,4. This occurs when the cells are moved from the incubator to the biosafety cabinet and/or microscope which may not be optimal for the cells. Previous studies have confirmed the advantages of culturing both pluripotent and neural stem cells in hypoxic conditions4,11, and for best results, these conditions need to be continuous. Moreover, risks of cellular contamination are higher as the laboratory environment and personnel impinge upon the cells at almost every step of their culture and manipulation. Traditional clean rooms comprise one effective method to greatly decrease contamination risks but they are expensive, have a large footprint and fail to address stressors related to carbon dioxide and oxygen concentrations. 
A cell production facility that can address both contamination risks and gas concentrations and that can be qualified to meet cGMP criteria9 provides high quality cells for basic science research as well as clinical applications1,6,7. Such a cell production facility consists, at a minimum, of the following components: a process chamber, which acts as a heated workspace for the feeding and manipulation of cell cultures; a laminar flow hood, for the initial sterilization of reagents, tubes, and tools; two buffering airlock chambers in between the hood and the process chamber; two cell culture incubators accessible from the process chamber; a microscope chamber adjacent to the process chamber; and finally, computer software to set and monitor the conditions within these modules. Using this basic infrastructure, a wide variety of tasks can be performed, such as standard feeding and passaging of pluripotent stem cells and multipotent neural stem cells, as well as more specialized methods like Sendai virus-based reprogramming, in vitro migration studies, and differentiation of neural stem cells for electrophysiological characterization.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;i&gt;Xvivo System&lt;/i&gt;</td><td>Biospherix</td><td>custom made</td><td></td></tr><tr><td>&lt;i&gt;Xvivo&lt;/i&gt; Software</td><td>Biospherix</td><td>version i.o.2.1.2.1</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>P-M2H-C3-S-U-OXY</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>M2H-C3-D-U-CO2</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Western Innovator</td><td>CTM75-7-2-2-BM</td><td></td></tr><tr><td>Microscope with DP21 camera and fluorescence</td><td>Olympus Corporation</td><td>CKX41</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12 Glutamax</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>StemPro hESC Supplement</td><td>Life Technologies</td><td>A100006-01</td><td></td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Phosphate-Buffered Sodium</td><td>Hyclone</td><td>9236</td><td></td></tr><tr><td>Fibroblast Growth Factor 2</td><td>R&amp;amp;D Systems</td><td>AFL233</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Protide</td><td>PP1130</td><td></td></tr><tr><td>Hank&#039;s-based Cell dissociation Buffer</td><td>Life Technologies</td><td>13150-016</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>R&amp;amp;D Systems</td><td>AFL236</td><td></td></tr><tr><td>Oct-3/4 Antibody</td><td>Millipore</td><td>AB3209</td><td></td></tr><tr><td>TRA-1-60 Antibody</td><td>Millipore</td><td>MAB4260</td><td></td></tr><tr><td>SSEA4 Antibody</td><td>Millipore</td><td>MAB4304</td><td></td></tr><tr><td>BIT-9500 Serum Supplement</td><td>Stemcell Technologies</td><td>9500</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumable Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 ml Serological pipet</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>5 ml Serological pipet</td><td>Olympus Plastics</td><td>12-102</td><td></td></tr><tr><td>10 ml Serological pipet</td><td>Olympus Plastics</td><td>12-104</td><td></td></tr><tr><td>25 ml Serological pipet</td><td>Olympus Plastics</td><td>12-106</td><td></td></tr><tr><td>50 ml Serological pipet</td><td>Olympus Plastics</td><td>12-107</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>353043</td><td></td></tr><tr><td>T25 flask</td><td>TPP</td><td>90026</td><td></td></tr><tr><td>T-75 flask</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>20 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491130</td><td></td></tr><tr><td>200 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491148</td><td></td></tr><tr><td>1,000 pipet tips</td><td>Eppendorf</td><td>22491156</td><td></td></tr><tr><td>Cryovials</td><td>Thermo Scientific</td><td>5000.102</td><td></td></tr><tr><td>70% ethanol</td><td>BDH</td><td>BDH1164-4LP</td><td></td></tr><tr><td>Sanimaster 4</td><td>Ecolab</td><td>65332960</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>A714239</td><td></td></tr></tbody></table>

culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Este manuscrito descreve com algum detalhe uma instalação de produção de células, e os aspectos únicos da cultura de células dentro de um sistema fechado. A maior parte das instalações de produção de células compreende um aparelho de cultura celular feito por encomenda, fechado, que tem uma pegada pequena e pode ser facilmente instalado dentro de um 6 x 7,5 m 2 quarto (Figura 2). Em nenhum momento são as células dentro do aparelho exposto ao pessoal de laboratório ou ambiente. O aparelho é constituído por vários módulos: uma câmara de processo, uma câmara de fluxo laminar, 2 câmaras de tamponamento Airlock entre o capuz e a câmara de processo, duas incubadoras de cultura de células, e um microscópio de câmara adjacente à câmara de processo (Figura 3). O sistema é controlado por software que permite variáveis ​​ambientais a serem controladas e monitoradas (Figura 1). O computador que executa o software está em uma fonte de alimentação ininterrupta. Os gases são alimentados em the sistema por um conjunto de colectores de oxigénio, azoto e dióxido de carbono (Figura 4). Gases para o dispositivo são fornecidos por sistemas múltiplos que têm dois conjuntos de tanques cada - um conjunto ativo e um conjunto de reserva. Quando o conjunto ativo é desenhada para baixo, o colector muda automaticamente para os tanques de substituição ordem reserva constituídos e de pessoal. O poder está em um sistema gerador de backup. Pluripotentes e células estaminais neurais podem ser cultivadas em condições de hipóxia nesta instalação utilizando protocolos previamente desenvolvidos 9, com as complicações adicionais inerentes ao equipamento novo. As células estaminais pluripotentes podem ser derivados utilizando vírus Sendai, utilizando anticorpos específicos de pluripotência para identificar colónias totalmente transfectadas, as quais são então isoladas e expandidas. (Figura 5A e B), A partir destes iPSCs, células estaminais neuronais e neurónios podem ser derivados usando SMAD inibição dupla, e o seu fenótipo verificado USI<html> Os anticorpos específicos para NSC ng (Figura 5C e D). <img alt="figura 1" src="/files/ftp_upload/53685/53685fig1.jpg" /> Figura 1. Interface Gráfica para o Mecanismo de célula de produção. (A) A representação gráfica do CPF é a tela padrão e combina o desenho CAD mostrado na Figura 3. Um clique do mouse sobre o buffer Módulo 1 (2F na Figura 1) abre seu ecrã de controlo (B), onde os valores de o 2 são definidas para coincidir com a câmara de processamento (3 na Figura 3). Da mesma forma, clicando sobre o Processo da Câmara ou Incubadora 1 traz suas respectivas telas de controle (C e D, respectivamente), onde os valores podem ser alterados ou monitorados, conforme apropriado.<html> rget = &quot;_ blank&quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 2" src="/files/ftp_upload/53685/53685fig2.jpg" /> Figura 2. O dispositivo de célula de produção. (A) O sistema como visto de uma vista frontal direita. Observe as conexões de alimentação e de gás no tecto e o carro com os acessórios de microscópio e computador para a direita. (B) A câmara de fluxo laminar com as portas de acesso aos módulos de tampão visto no lado direito. (C) A câmara de processo com as incubadoras duas portas (preto) visto na parte traseira. (D) Uma vista através do lado direito do sistema que mostra o microscópio e monitor com as portas para as câmaras de amortecimento visto à distância. <a href="https://www.jove.com/files/ftp_upload/53685/53685fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> t &quot;fo: manter-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/53685/53685fig3.jpg" /> Figura 3. Um desenho de CAD do mecanismo de célula de produção. Todos os artigos que entram no dispositivo são primeiramente limpo com álcool e seco ao ar na câmara de fluxo laminar (1). Os artigos são, em seguida, transferida para o ambiente apropriado no módulo de tampão dianteira (2F) antes de serem passados ​​para o módulo de UCPC, ou processo de câmara (3). As células são cultivadas nas duas incubadoras (4, 5). Coloração de células vivas, colônia colheita, a avaliação da morfologia celular geral e análise de migração ocorre no módulo UPC, ou Microscópio Câmara (6). Finalmente, resíduos sai do sistema através do módulo tampão traseiro (2R). <a href="https://www.jove.com/files/ftp_upload/53685/53685fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> &lt;<html> / P&gt; <img alt="Figura 4" src="/files/ftp_upload/53685/53685fig4.jpg" /> Figura 4. Fontes de Gases e poder. (A) O colector de CO 2 é uma configuração de 4 x 4, uma vez que fornece todos os incubadoras de laboratório. (B) O colector de O 2 é uma configuração única e suprimentos a instalação de produção de células de 2 x 2. Azoto para o sistema é gerado num vaporizador (C, apenas o sinal de saída abaixo) ligado a um colector de azoto líquido (à direita) que também enche automaticamente cryofreezers (inferior esquerdo). O colector é uma configuração 2 x 2 sendo fornecida por dois pares de tanques de 160 L de azoto líquido. CO 2, O 2, N 2 e são fornecidos através do teto shutoffs (D). câmara de vácuo é também fornecida neste local. Visto apenas atrás das shutoffs gás de alimentação são um par de cabos de energia que fornecem energia ao sistema.f = &quot;https://www.jove.com/files/ftp_upload/53685/53685fig4large.jpg&quot; target = &quot;_ blank&quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 5" src="/files/ftp_upload/53685/53685fig5.jpg" /> Figura 5. As células pluripotentes estaminais derivadas em hipóxia com o vírus Sendai. (A) SC187-SF4-2I0-E3 iPSCs iPSCs (nomenclatura encontrados em Stover et al. 9), contraste de fase 10x. (B) SC88.1-UH1-2I0 iPSCs live-coradas com Tra-1-60 AF-594-rotulados, diluição 1: 100 na mídia. Todos os iPSCs foram transduzidas na instalação de produção de células em 5% de O 2 com o vírus de Sendai. (C) O contraste de fase de NSC-SC68.1 UH0-2I0-M0S13-N2G6 iPSC-derivados, crescido em 5% de O 2 em lâminas de câmara. As células foram então fixadas em paraformaldeído a 4% e coradas com o anticorpo primário anti-nestina humana, e o anticorpo secundário Alex-488. Todas as barras de escala são a 1001;. M <a href="https://www.jove.com/files/ftp_upload/53685/53685fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Cultura pluripotentes humanas e células-tronco neurais em um sistema de cultura celular fechado de Pesquisa Básica e pré-clínica

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

culturing-human-pluripotent-neural-stem-cells-an-enclosed-cell

Research

JoVE Journal

Developmental Biology

10.2K visualizações.  Childrens Hospital of Orange County Research Institute. Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Vídeo: Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

A Cultura Modelo Novel para pluripotentes estaminais humanas Propagação celular em gelatina no Media Placenta condicionado

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Biologia do Desenvolvimento

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Passaging células-tronco neurais

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Biologia

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

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 ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Diferenciação neural eficiente usando a cultura unicelular de células-tronco embrionárias humanas

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Diferenciação de células-tronco pluripotentes induzidas usando um sistema de cultura automatizado

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Autor em destaque: Automatizando a cultura iPSC para maior reprodutibilidade 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

Um Sistema de Cultura Automatizado para Manutenção e Diferenciação de Células-Tronco Pluripotentes Induzidas por Humanos

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para pluripotentes estaminais humanas produção de células, Manutenção e Análise Genética

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Culturas de células mistas de cérebro de camundongo embrionário para estudar infecção e imunidade inata

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Autor em destaque: Estabelecendo culturas mistas de células neuronais e gliais de cérebros embrionários de camundongos para estudar infecções e imunidade inata  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Estabelecendo culturas mistas de células neuronais e gliais de cérebros embrionários de camundongos para estudar infecção e imunidade inata

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

Cultura pluripotentes humanas e células-tronco neurais em um sistema de cultura celular fechado de Pesquisa Básica e pré-clínica

Resumo

Explore mais vídeos

PLAYLIST