Esta pesquisa é apoiada pela Fundação Hartwell, a American Heart Association (Scientist Desenvolvimento Grant # 0830384N) ea Fundação Leducq: 07CVD04 Mitral.

1. Preparação <ol><li> Incubar codorna fértil ou ovos de galinha para a fase 14 - (cerca de 2 dias) ou 25 (cerca de 4,5 dias) em 60% de umidade e 37 ° C. </li><li> Preparar a solução de Earl estéril de salina balanceada (EBSS) <ol><li> Adicionar 100 mL EBSS 10X a 600 mL de água 18 mohms </li><li> Adicionar 2,2 g de bicarbonato de sódio </li><li> Ajustar o pH da solução para 7,2 </li><li> Levar a solução até 1000 mL </li><li> Esterilizar a solução, passando por um filtro de 0,2 mM </li></ol></li><li> Prepare meio de cultura estéril M199. <ol><li> Adicione 1 pacote de pó para M199 700 mL de água 18 mohms. </li><li> Adicionar 2,2 g de bicarbonato de sódio </li><li> Ajustar o pH da solução para 7,2. </li><li> Adicionar 10 mL (1%) Penicilina-estreptomicina </li><li> Levar a solução até 1000 mL </li><li> Esterilizar a solução, passando por um filtro de 0,2 mM </li><li> Adicionar 1 mL estéreis suplemento Insulin-transferrina-selênio-G 100X </li><li> Adicionar 10 mL (1%) de soro de galinha estéril </li></ol></li><li> Prepare três géis de colágeno dimensional em uma concentração de colágeno de 1,5 mg / mL. Trabalho anterior em nosso laboratório demonstraram que a concentração de colágeno de mais de 1,5 mg / mL fornece uma matriz que é muito dura biomecanicamente para que as células se movem através. As concentrações de gel de colágeno inferior a 1,5 mg / mL têm fibras colágenas tão poucas que a touceira de células pode rasgar as fibras localmente, produzindo uma ilha de células. <ol><li> Adicione o seguinte gelada reagentes a um tubo de centrifugação estéril 15 mL em ordem: <ul><li> 3X M199: Volume = volume total necessário / 3 </li><li> Estéril 18 mohms água: Volume = volume total necessário (M199 volume + volume de frango de soro + 0,1 M NaOH volume de volume de colágeno +) </li><li> Soro de galinha estéril: Volume = volume total necessário * 0,01 </li><li> Colágeno de ratos de cauda I: Volume = (concentração do gel de colágeno * volume total necessário) / concentração de colágeno estoque </li><li> Estéril 0,1 M NaOH: Volume = volume de colágeno * 0,2 Nota: A quantidade de água, 0,1 M NaOH, e colágeno adicionado irá variar dependendo da concentração da solução de colágeno. </li></ul></li><li> Misturar a solução de colágeno bem com uma pipeta estéril. </li><li> Transferir imediatamente 0,3 mL da solução de colágeno em 1,9 centímetros 2 poços área de crescimento. Esta é a solução de colágeno suficiente para cobrir completamente o bem e para fornecer um gel que é de aproximadamente 3 mm de espessura. A espessura do gel de pelo menos 250 M é necessário para os estudos invasão celular. </li><li> Permitir que a solução de colágeno para gel por pelo menos 1 hora a 37 ° C, 5% CO 2, e 100% de umidade. </li></ol></li></ol> 2. Pré-EMT isolamento de células Endocárdica e Cultura <ol><li> Isolamento de embriões <ol><li> Coloque um pedaço de fita de polegada de comprimento de laboratório sobre a extremidade maior do ovo. Este é o lugar onde a célula de ar está localizado. A fita estabiliza a casca de ovo de codorna frágil. </li><li> Delicadamente fure a área gravada do ovo na célula de ar com a ponta da tesoura esterilizada curvas. </li><li> Corte um buraco na parte gravada do ovo que é grande o suficiente para permitir a gema para passar. Iniciar o buraco na punção e cortar para fora em forma de espiral. Certifique-se de cortar a membrana externa, mas evitar perfurar a gema. </li><li> Quando o buraco no casco está completo, visualizar o embrião de codorna. O embrião é, normalmente localizados na parte superior da gema. </li><li> Começará a derramar a gema através do buraco cortado no ovo com uma mão. Segure uma pinça # 55 na outra mão. Como o embrião sai do ovo na parte superior da gema, deslize suavemente as pinças # 55 sob o embrião e removê-lo da gema. Nota: Para ovos de galinha, o isolamento de embriões envolve quebrar o ovo de galinha em uma superfície limpa e nítida quebra o ovo em um prato de 100 milímetros Petri. O embrião deve estar localizado na parte superior da gema. Deslize suavemente # 55 pinças sob o embrião e removê-lo da gema. </li><li> Coloque o embrião em um prato de 100 milímetros de Petri contendo gelada, EBSS estéril. </li></ol></li><li> Estágio os embriões de acordo com os critérios de Hamburger e Hamilton 1. Embriões no estádio 14 - são palco últimos 13, mas ainda não no estágio 15, com 20-21 somitos e um broto de cauda. Uma característica fundamental do estágio 14 - embriões é o ângulo de cabeça, que é ligeiramente maior do que 90 ° para o corpo. </li><li> Remove os corações de embriões de codorna na fase 14 - com # 55 pinças cortando transversalmente onde o canal AV e OFT fazer contato com a parede torácica. Coloque os corações juntos em um lado da placa de Petri. </li><li> Recolher os corações isolados com uma pipeta de transferência estéril e colocá-los em um 100 milímetros nova placa de Petri cheia de estéril, gelada EBSS. </li><html><li> Corte o OFTs e canais AV transversalmente dos ventrículos com # 55 pinças. </li><li> Seção dos canais restantes AV e / ou vias de saída (OFTs) longitudinalmente. </li><li> Definir uma pipeta para um volume de 20 uL. Utilize este pipeta para aspirar seis longitudinalmente canais de corte e OFTs AV. </li><li> Expulsar os canais AV e OFTs para o gel de colágeno. Aspirar EBSS qualquer excesso da superfície do gel. </li><li> Use # 55 pinças para orientar os explantes que eles são lisos e assim o lado da luz enfrenta o gel de colágeno. Tente não perfurar o gel de colágeno com a pinça. </li><li> Depois de 2 horas a 37 ° C e 5% CO 2, remova o miocárdio dos explantes com # 55 pinças. As células endocárdicas deve ser deixado para trás na superfície do gel de colágeno. Alternativamente, o miocárdio pode ser deixado no explante. Com a retirada do miocárdio, as células endocárdicas não sofrer com EMT sem intervenção. Com o miocárdio apresentam um subconjunto de células endocárdicas vai se transformar em mesênquima, mas o grau de transformação pode ser modulada com fatores externos. Nota: Mesmo que o miocárdio será deixado ligado, o explante deve ser permitido, pelo menos, duas horas para anexar ao gel antes média é adicionado. </li><li> Adicionar 0,4 mL M199 médio a cada explantes contendo bem. </li><li> Cultura das células a 37 ° C, 5% CO 2, e 100% de umidade. </li></ol> 3. Pós-EMT Almofada AV isolamento de células mesenquimais e Cultura <ol><li> Rachar o ovo de galinha em uma superfície limpa e nítida quebrar o ovo em um prato de 100 milímetros Petri. O embrião deve estar localizado na parte superior da gema. Pegue o embrião com # 5 pinças agarrando as membranas que envolvem o embrião e, em seguida, coloque o embrião para uma placa de Petri 100 milímetros enchido com o gelo frio, EBSS estéril. </li><li> Estágio os embriões de acordo com os critérios de Hamburger e Hamilton 1. Características de um embrião de estágio 25 incluem cotovelo distintas e articulações do joelho e pigmentação nos olhos. </li><li> Isolar os corações de todos os embriões HH25 e colocar o coração juntos em um lado da placa de Petri. </li><li> Recolher os corações isolados com uma pipeta de transferência estéril e colocá-los em um 100 milímetros nova placa de Petri cheia de estéril, gelada EBSS. </li><li> Isolar a região da AV todos os corações de uma vez. Coloque o AVs juntos em um lado da placa de Petri. Agite suavemente a região AV para remover todo o sangue, que podem afetar a viabilidade celular. </li><li> Recolher os AVs isolada com uma pipeta de transferência estéril e colocá-los em um 100 milímetros nova placa de Petri cheia de estéril, gelada EBSS. </li><li> Seqüencialmente isolar as almofadas da AV. Certifique-se que não há nenhum presente miocárdio nas almofadas AV. </li><li> Recolher as almofadas isolada com uma pipeta de transferência estéril e colocá-los em um tubo de centrifugação estéril 15 mL. </li><li> Girar as almofadas para o fundo do tubo de centrífuga de 15 xg por 3 minutos. </li><li> Use uma ponta mL estéril 1000 para remover o máximo do sobrenadante EBSS possível, sem perturbar as almofadas. </li><li> Adicionar 1 mL de 0,25% Tripsina-EDTA que foi pré-aquecido a 37 ° C. Permitir que as almofadas para incubar em tripsina até que estejam completamente dissolvidos, que é normalmente cerca de 3-5 minutos. </li><li> Adicionar 100 mL de soro de frango para o tubo de centrifugação para saciar a tripsina. </li><li> Agregar as células a 160 xg por 5 minutos. Remover o sobrenadante com uma estéril, ponta 1.000 mL. </li><li> Ressuspender as células em 2 mL de M199 médio e misture por pipetagem. Tomar 10 mL da suspensão de células de contar com hemocitômetro e determinar o número total de células que foram isolados </li><li> Determinar quantos géis de colágeno será feita com base no número de células isoladas. Células precisam ser banhado a uma densidade de 2 x 10 5 células / mL com um volume de 250 L gel. </li><li> Agregar as células a 160 xg por 5 minutos. Remover o sobrenadante com uma estéril, ponta 1.000 mL. </li><li> Usando o protocolo listados acima, adicione 3X M199, água, soro de galinha, o colágeno, e 0,1 M NaOH, nessa ordem, para o pellet celular. Mix pipetando gentil. </li><li> Adicionar 250 mL para cada 1,9 cm 2 área de crescimento bem. </li><li> Permitir que a solução de colágeno para gel por pelo menos 1 hora a 37 ° C, 5% CO 2, e 100% de umidade. </li><li> Adicionar 0,4 mL de meios de cultura e incubar durante a noite. </li><li> Para continuar a cultivar os géis em condições livre de estresse, liberar os géis dos lados da cultura bem usando uma ponteira estéril 200 mL. Culturas mecanicamente restrito são géis que permanecem ligados para os lados da cultura também. </li></ol> 4. Resultados representante <img src="/files/ftp_upload/2159/2159fig1.jpg" alt="Figura 1" /> Figura 1:. Exemplos de embriões (A) embrião HH14-codorna, e (B) embrião de galinha HH25 são mostrados aqui. fig2.jpg &quot;alt =&quot; Figura 2 &quot;/&gt; Figura 2: HH14 válvula de explantes endocárdica após 2 horas de cultura (A) com o presente do miocárdio, ou (B) com o miocárdio removido.. <img src="/files/ftp_upload/2159/2159fig3.jpg" alt="Figura 3" /> Figura 3:. HH14 válvula de explantes endocárdica (A) Com o presente miocárdio, muitas das células sofrem endocárdica para a transformação mesenquimais e ter um fusiformes fenótipo, mesenquimais após 48 horas de cultura. (B) Quando o miocárdio é removido todas as células manter uma calçada em forma de fenótipo, endocárdio após 48 horas de cultura. <img src="/files/ftp_upload/2159/2159fig4.jpg" alt="Figura 4"/> Figura 4:. Cells HH25 células mesenquimais em uma válvula de gel de colágeno são arredondados imediatamente após a semeadura. <img src="/files/ftp_upload/2159/2159fig5.jpg" alt="Figura 5"/> Figura 5:. HH25 células mesenquimais em cultura (A) Após 48 horas de cultura em um gel de colágeno restringiu o HH25 células mesenquimais estão começando a se espalhar. (B) HH25 células em um gel de colágeno sem stress 7 dias após a semeadura e 6 dias depois da libertação ter compactado o gel de aproximadamente 50% da área original.

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li></ol>

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O método para isolar células endocárdicas do estágio HH14 - corações originalmente desenvolvido pela Runyan e Markwald fornece um ambiente controlado, in vitro ambiente para estudar os fatores que iniciam e modulam EMT embrionárias 2. HH14 - explantes cultivados endocárdica sem miocárdio não sofrerá EMT sem intervenção biomecânica ou bioquímica. Se o miocárdio é deixado no explante será sinal de um subconjunto de células endocárdicas submeter-se a transforma?...

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<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
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<td>Dumont tweezers #55</td>
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<td>World Precision Instruments, Inc.</td>
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<td>Dumont tweezers #5</td>
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<td>World Precision Instruments, Inc.</td>
<td>14098</td>
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<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
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<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
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<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
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<td>Sterile 15 mL centrifuge tubes</td>
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<td>Corning</td>
<td>430828</td>
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<td>Sterile petri dishes, 100 mm x 15 mm</td>
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<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
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<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
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<td>Rat-tail collagen I</td>
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<td>10X Earl's Balanced Salt Solution</td>
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<td>M199 powder</td>
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<td>Invitrogen</td>
<td>31100-035</td>
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<td>Penicillin-Streptomycin</td>
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<td>Invitrogen</td>
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<td>Invitrogen</td>
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<td>Invitrogen</td>
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<td>Sodium hydroxide solution, 1.0 N</td>
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<td>Sigma-Aldrich</td>
<td>S2770</td>
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<td>Fertile quail eggs (Coturnix coturnix)</td>
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<td>Lake Cumberland Game Bird Farm</td>
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<td>Fertile chicken eggs (Gallus gallus)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
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isolation and culture of avian embryonic valvular progenitor cells

Adequada formação e função das válvulas cardíacas embrionárias é fundamental para a progressão de desenvolvimento. O coração embrionário é um tubo em forma de U do endocárdio cercado por miocárdio. O miocárdio secreta cardíaca geléia, um ácido hialurônico rico em matriz gelatinosa, na junção atrioventricular (AV) e via de saída (OFT) lúmen. No estágio HH14 valvulogênese começa quando um subconjunto de células endocárdicas receber sinais do miocárdio, submetidos à transformação do endocárdio mesenquimais (EMT), e invadir a geléia cardíaca. No estágio HH25 as almofadas valvular são totalmente mesenchymalized, e é esta mesênquima que, eventualmente, as formas do aparelho valvar e septal do coração. Compreensão dos mecanismos que iniciam e modulam o processo de diferenciação celular e EMT são importantes por causa de sua conexão com graves defeitos cardíacos congênitos. Neste estudo, apresentamos métodos para isolar pré-EMT endocárdicas e células mesenquimais pós-EMT, que são os dois fenótipos celulares diferentes da almofada prevalvular. Células pré-EMT endocárdica podem ser cultivadas com ou sem o miocárdio. Pós-EMT células mesenquimais almofada AV podem ser cultivadas géis de colágeno dentro mecanicamente restrito ou livre de estresse. Estas 3D em modelos in vitro imitar os principais eventos da morfogênese valvular e são úteis para desconstruir os mecanismos de valvulogênese estágio precoce e tardia.

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Este artigo irá fornecer um método para isolar e cultura de codorna ou frango HH14<sup&gt; -</sup&gt; Células de válvulas do endocárdio e HH25 células mesenquimais válvula de amortecimento.

Isolamento e Cultura da Avian embrionárias Células Progenitoras Valvular

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

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Biology

10.1K visualizações.  Cornell University. Este artigo irá fornecer um método para isolar e cultura de codorna ou frango HH14 - Células de válvulas do endocárdio e HH25 células mesenquimais válvula de amortecimento.

Vídeo: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

Isolamento e Cultura de Pós-Natal do rato Cerebelar Granule Células Progenitoras Neuron e Neurônios

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Biologia

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 ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e expansão em grande escala de Adulto Humanos endotelial Colony Células Progenitoras Forming

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultas e embrionárias Cultura Microexplant Músculo Esquelético e isolamento de células-tronco musculares

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Isolamento de Valvular Células Endoteliais

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e cultura de células da zona nefrogênica do rim do rato embrionárias

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e cultura primária de células hepáticas de ratos

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolamento de Células Embrionárias ventriculares endoteliais

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...