Nota: Para manter as condições de cultura estéril, todos os procedimentos neste protocolo são realizadas utilizando práticas de laboratório estéril e conduzidos sob uma capela de fluxo laminar. Antes de começar, certifique-se que qualquer mídia é equilibrado a 37 ° C e adequadamente gaseados. Preparar pratos Geltrex revestido Cultura Nota: ver anexo para o uso de placas de cultura de CELLstart revestido <ol><li> Tubo de descongelar um dos Geltrex (1 mL) lentamente a 2-8 ° C e diluir 1:100 em 99 mL de Knockout D-MEM/F-12. Misturar a solução suavemente. Nota: Algumas linhagens de células iPS podem requerer uma diluição Geltrex diferentes para o crescimento ideal. Veja o apêndice para diluições de alternativa. </li><li> Cobrir toda a superfície de cada placa de cultura com a solução Geltrex (1 mL para um prato de 35 mm, 1,5 mL para um prato de 60 mm). </li><li> Seal cada prato com Parafilm para evitar a secagem e incubar os pratos para 1 hora a 37 ° C. Nota: Neste ponto, você pode armazenar os pratos Geltrex revestido cultura a 4 ° C por até 1 mês. Seal cada prato com Parafilm para impedir a Geltrex sequem. </li><li> Antes de usar, transferir os pratos Geltrex revestido a uma capela de fluxo laminar e permitir-lhes atingir a temperatura ambiente (cerca de 1 hora). </li></ol> Preparando Alimentador-Free SR Completa KnockOut Médio <ol><li> Para preparar 1 mL de 10 mcg / ml solução FGF Basic, assepticamente misturar os componentes listados abaixo. Alíquota da solução e armazenar a -20 ° C por até 6 meses. <table border="0"><tbody><tr><td> FGF básico </td><td> 10 mg </td></tr><tr><td> D-PBS </td><td> 990 mL </td></tr><tr><td> 10% BSA </td><td> 10 L </td></tr></tbody></table> Nota: BSA pode ser substituído com HSA ou SR Knockout na mesma concentração. </li><li> Para preparar 50 mL de solução 2 Dispase mg / mL, assepticamente misturar os componentes listados abaixo. Filtro de esterilizar a solução e armazenar a 4 ° C por até duas semanas. <table border="0"><tbody><tr><td> Dispase </td><td> 100 mg </td></tr><tr><td> D-PBS </td><td> 50 mL </td></tr></tbody></table></li><li> Para preparar 100 mL de SR KnockOut completa Alimentador-Free (KSR-FF) médio assepticamente combinar os componentes listados na tabela abaixo. <table border="1"><tbody><tr><td> Componente </td><td> Concentração de ações </td><td> Concentração Final </td><td> Volume </td></tr><tr><td> Knockout DMEM/F12 (Cat. n º. 12.660-012) </td><td> - </td><td> 1X </td><td> 76,8 mL </td></tr><tr><td> Glutamax-I (Cat. No. 35050-061) </td><td> 200 mM </td><td> 2 mM </td><td> 1 mL </td></tr><tr><td> KnockOut SR (sem Cat.. 10.828-028) </td><td> - </td><td> 20% </td><td> 20 mL </td></tr><tr><td> KnockOut SR-GFC (sem Cat.. A10580-01) </td><td> 50X </td><td> 1X </td><td> 2 mL </td></tr><tr><td> bFGF (sem Cat.. PHG0024) </td><td> 10 mcg / mL </td><td> 20 ng / mL </td><td> 200 mL </td></tr></tbody></table> Você pode armazenar o meio KSR-FF a 2-8 ° C por até uma semana. </li><li> Pouco antes de pré-equilibrar o meio completo à temperatura e gases, adicionar assepticamente o volume necessário de 2-mercaptoetanol (55 mM de concentração de ações) para uma concentração de 0,1 mM final. Por exemplo, para preparar 100 mL da KSR-FF médio mL adicionar 182 de 55 mM 2-mercaptoetanol (1:550 diluição) Alternativamente, o 2-mercaptoetanol podem ser adicionadas ao meio 1X preenchido e armazenado a 2-8 ° C para até uma semana. </li></ol> Adaptação iPSCs a KSR-FF Médio <ol><li> Antes de começar, pré-equilibrar os seus pratos Geltrex revestidas até a temperatura ambiente no bairro. </li><li> Cultura da iPSCs em células alimentadoras MEF até que sejam confluentes 70-80%. Por favor, consulte o manual Fibroblasto GIBCO embrionárias de camundongos para protocolos baseados em MEF cultura. </li&gt; <li> Pré-aquecer o volume necessário de Dispase em um banho de água 37 ° C. Consulte a Tabela 1 abaixo para obter detalhes sobre os volumes necessários. </li><li> Pré-equilibrar o volume necessário de KSR-FF em um banho de água a 37 ° C for15 min. Consulte a Tabela 1 abaixo para obter detalhes sobre os volumes necessários. </li><li> Aspirar o meio dos pratos, cultura e adicionar uma quantidade adequada de solução Dispase. Incubar os pratos a 37 ° C por 3-5 minutos. </li><li> Aspirar a solução de cada navio Dispase cultura e lavar as células alimentador MEF suavemente com D-PBS (2 a 3 vezes). </li><li> Adicionar uma quantidade adequada de meio completo KnockOut SR a cada navio de cultura. Use um raspador de célula ou uma pipeta de 5 mL para raspar suavemente as células da superfície da embarcação cultura. Nota: veja o apêndice para protocolos alternativos para adaptação difícil de se adaptar as linhas de células iPS, </li><li> Recolher a suspensão de células de cada placa de cultura em diferentes tubos de 15 mL cônico. Lave cada navio cultura com uma quantidade adequada de meio completo KnockOut SR, e adicione a D-PBS lavar médio para o 15 mL tubos cônicos contendo a suspensão celular. Seja cauteloso para não quebrar os aglomerados de células em uma única célula </li><li> Centrifugar a 15 mL em tubos cônicos 200 xg por 5 minutos para sedimentar as iPSCs. </li><li> Aspirar o sobrenadante do pellet iPSC. Ressuspender o sedimento em uma quantidade adequada de KSR-FF médio de acordo com a taxa de divisão (Tabela 1). Não quebre os aglomerados de células para um tamanho menor, porque os aglomerados mais pequenos não dão bem para a superfície. Nota: Recomenda-se uma taxa de divisão de 1:2 para os primeiros três passagens após o iPSCs foram várias passagens diretamente do Meio de Cultura iPSC MEF para KSR-FF médio. Normalmente, a razão de separação 01:03 - 01:05 é apropriado, mas passaging em 01:02 garante a maior densidade de células necessárias ao adaptar em uma cultura de alimentação livre. </li><li> Antes de chapeamento do iPSCs em pratos Geltrex revestido, aspirar solução Geltrex residual do prato pré-revestidos, e lentamente adicionar uma quantidade adequada de suspensão de células de cada placa de cultura. Nota: não lavar os pratos antes de chapeamento. </li><li> Mover a placa de cultura e para trás e para os lados várias vezes para dispersar as células em toda a superfície do prato. Delicadamente, coloque o prato de cultura em uma incubadora de 37 ° C com uma atmosfera húmida de 4 a 6% CO 2 no ar. Substituir o médio gasto com KSR-FF a cada dia. </li></ol> Passaging células iPS usando KSR-FF <ol start="13"><li> Observar o iPSCs humana crescendo em completa KSR-FF sob o microscópio para confirmar que as células são 70-80% confluentes e pronto para ser repicadas. Consulte a Figura 1. Nota: Se colônias tornam-se muito denso ou muito grande, maior diferenciação ocorre. </li><li> Cortar e remover qualquer colônias diferenciada iPSC antes passaging a cultura. </li><li> Pré-aquecer o volume necessário de Dispase em um banho de água 37 ° C. Consulte a Tabela 1 abaixo para obter detalhes sobre os volumes necessários. </li><li> Pré-equilibrar o volume necessário de KSR-FF em um banho de água a 37 ° C for15 min. Consulte a Tabela 1 abaixo para obter detalhes sobre os volumes necessários. </li><li> Aspirar o meio de gastos do navio cultura usando uma pipeta, e lavar as células duas vezes com D-PBS. </li><li> Delicadamente adicionar pré-aquecido Dispase solução para o navio de cultura (por exemplo, 1 mL de solução Dispase por 60 mm placa de cultura). Redemoinho do navio cultura para revestir a superfície da célula inteira. </li><li> Incubar a embarcação cultura a 37 ° C por 3 minutos. </li><li> Remover o navio da incubadora, aspire a solução Dispase e lavar cuidadosamente as células com D-PBS. </li><li> Raspe as células da superfície da placa de cultura com uma espátula de células, e transferência das células para um tubo de centrifugação estéril 15ml. </li><li> Lavar o prato de cultura duas vezes com KSR-FF, gentilmente &quot;pulverização off&quot; as células que não tenham destacado. Piscina do enxágüe meio com as células no tubo de 15mL. </li><li> Centrifugar o tubo a 200 xg por 5 minutos em temperatura ambiente para agregar as células. </li><li> Aspirar cuidadosamente o sobrenadante sem perturbar o pellet celular e descartá-lo. </li><li> Agite suavemente o tubo para expulsar totalmente o pellet celular a partir do fundo do tubo. </li><li> Delicadamente ressuspender as células pré-equilibradas KSR-FF com uma pipeta de 5mL sorológicos. Não triturar. Nota: é fundamental na etapa delicadamente ressuspender as células sem usar a força para evitar danos. </li><li> Transferência das células para um novo prato de 60 mm Geltrex revestido na razão de separação desejada e mova a placa de cultura e para trás e para os lados várias vezes para dispersar as células em sua superfície. </li><li> Coloque o prato de cultura em uma incubadora de 37 ° C com uma atmosfera húmida de4 a 6% CO 2 no ar. </li><li> No dia seguinte, colocar suavemente o médio gasto com KSR-FF para remover restos celulares. Substituir o médio passou todos os dias depois. Observe as células iPS diárias e passagem-los conforme necessário (aproximadamente a cada 4-5 dias). Passaging é recomendada quando as células atingem confluência de 70-80%. Para criopreservação de células iPS e descongelamento, consulte nosso protocolo intitulado &quot;criopreservação e recuperação das células humanas iPS usando Completa KnockOut Feeder livre de substituição de soro Medium&quot;. </li></ol> Resultados esperados <img src="/files/ftp_upload/2236/2236fig1.jpg" alt="Figura 1" /> Figura 1. A imagem abaixo mostra contraste de fase iPSCs cultivadas em pratos Geltrex revestido cultura usando completa KnockOut Alimentador-Free substituição Serum médio. O iPSCs apresentam morfologia semelhante ao hESCs, caracterizado por núcleos grandes e citoplasma escasso. (40x) <table border="1"><tbody><tr><td> Componente </td><td> Dish 35 milímetros </td><td> Dish 60 milímetros </td><td> Dish 100 milímetros </td></tr><tr valign="top"><td> Completa KnockOut SR médio </td><td> 2 mL </td><td> 4 mL </td><td> 10 mL </td></tr><tr valign="top"><td> Geltrex Solution </td><td> 1 mL </td><td> 1,5 mL </td><td> 4-5 mL </td></tr><tr valign="top"><td> Dispase </td><td> 0,5 mL </td><td> 1 mL </td><td> ML 3-4 </td></tr><tr valign="top"><td> D-PBS para lavagem </td><td> 2 mL </td><td> 4 mL </td><td> 10 mL </td></tr></tbody></table> Tabela 1. Volumes recomendado <a href="http://www.jove.com/files/ftp_upload/2236/2236appendix.pdf">Por favor, clique aqui para ver o apêndice</a> .

<ol>
	<li>Takahashi, K. <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+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K. <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+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J. <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+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Li, W. <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+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Liao, J. <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+induced+pluripotent+stem+cell+lines+from+adult+rat+cells.">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li><li>Dimos, J. T. <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+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li><li>Park, I. H. <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+human-induced+pluripotent+stem+cells.">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li></ol>

Os autores deste artigo são empregados por Life Technologies, que produz reagentes e instrumentos utilizados neste artigo.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 12660-012</td>
<td>Note: see appendix for the use of alternative DMEM products</td>
</tr>
<tr>
<td>GlutaMAX-I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. No. 35050-061</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR, Cat. no. 10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR-GFC, Cat. no. A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>bFGF, Cat. no. PHG0024</td>
<td>Note: see appendix for alternative bFGF pack sizes.</td>
</tr>
<tr>
<td>2-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 17105-041</td>
<td>Note: see appendix for alternative passaging methods.</td>
</tr>
<tr>
<td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. A10480-01</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Tissue Culture Hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette-Aid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile serological pipettes (5 mL, 10 mL)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium

A descoberta em 2006 que fibroblastos humanos e mouse poderia ser reprogramado para gerar células iPS 1-3 com qualidades muito semelhantes às células-tronco embrionárias criou uma valiosa fonte de novas células pluripotentes para descoberta de medicamentos, terapia celular, e pesquisa básica. Media GIBCO e reagentes têm estado na vanguarda da pesquisa com células-tronco pluripotentes por anos. Knockout DMEM suplementado com Knockout Serum substituição é a mídia de sua escolha para o crescimento de células-tronco embrionárias e, agora, de cultura de células iPS 3-9. Este padrão ouro sistema de mídia agora pode ser usado para alimentação livre de cultura com a adição de Knockout SR Cocktail Fator de Crescimento. ES humana e métodos tradicionais de cultura de células iPS requerem o uso de mouse ou camadas de alimentação humana de fibroblastos, ou alimentador condicionado médio. Estes métodos cultura são de trabalho intensivo, difícil de escala e é difícil manter quadris células indiferenciadas, devido às condições indefinido. Invitrogen desenvolveu Knockout SR Cocktail Fator de Crescimento para permitir que você facilmente a transição do seu celular para culturas quadris alimentador livre, mantendo o uso do SR Knockout.

Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

O protocolo a seguir fornece instruções para a adaptação humana tronco pluripotentes induzidas (iPS) células para alimentação livre de cultura usando completa KnockOut Feeder livre de substituição de soro médio (KSR-FF). Uma vez adaptado, instruções para a manutenção contínua também são fornecidos.

Alimentador livre adaptação, Cultura e Passaging de células iPS humanas usando Completa KnockOut Alimentador-Free substituição Serum Médio

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

feeder-free-adaptation-culture-passaging-human-ips-cells-using

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Biology

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Vídeo: Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

MATIZ Passaging-tronco embrionárias humanas Cell-linhas com Tripsina

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Biologia

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Células ES: Cultura A partir de células congeladas

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

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 ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e Manutenção de células estaminais embrionárias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Criopreservação e recuperação das células humanas iPS usando KnockOut Completa Feeder livre de substituição de soro Médio

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Geração de cardiomiócitos Humanos: Um Protocolo de Diferenciação de Feeder livres Humanos células-tronco pluripotentes induzidas

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Isolamento de ARN a partir de pequenas não-codificante de Soro Humano

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

Perfis de expressão de microRNA iPS Human Cells, Retinal Pigment Epithelium Derivado de Ips, e Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Eficiente Geração células iPS a partir de sangue, utilizando epissomas e HDAC Inibidores

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Análise comparativa da hormona de crescimento humano no soro utilizando SPRI, Nano-SPRI e ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...