Agradecemos ao Dr. Shahragim Tajbakhsh para a prestação de camundongos Myf5 + / nLacZ. Agradecemos também a Alexander Hron e Michael Baumrucker para a leitura crítica do manuscrito. Este trabalho foi financiado por doações da Associação de Distrofia Muscular (MDA) e Gregory Marzolf Jr. MD Centro Award.

Os animais foram alojados em um ambiente SPF e foram monitorados pela pesquisa Recursos Animais (RAR), da Universidade de Minnesota. . Os animais foram sacrificados através de meios adequados (CO 2 inalação ou injecção de KCl, após ter sido anestesiados com injecção IP de Avertin (250 mg / kg) Todos os protocolos foram aprovados pelo Animal Care and Use Committee &#39;da Institucional (IACUC, Número Código: 1304-30.492 ) da Universidade de Minnesota. 1. Isolamento de células mononucleares de Rato Músculo Esquelético <ol><li> Devidamente sacrificar um ou dois ratos adultos jovens (3-8 semanas). <ol><li> Aperte e cortou a pele do abdômen com uma tesoura afiada. Retire a pele para mostrar completamente tríceps e musculatura dos membros posteriores (puxar a pele em direções opostas). </li><li> Remova todos os músculos das pernas esqueléticas (tibial anterior, gastrocnêmio e quadríceps) e tríceps ao longo dos ossos com uma tesoura. Em seguida, transferir os músculos para gelado, PBS estéril numa placa de 10 centímetros. </li></ol></li><li> Lave sangue de músculos em PBS e músculos de transferência para um novo 6 centímetros estéril placa: 1 placa de 1-2 ratos. </li><li> Remover o tecido conjuntivo, vasos sanguíneos, feixes nervosos e tecido adipogênica sob um microscópio de dissecação. </li><li> Com uma tesoura para oftalmologia, cortar e picar o tecido em uma pasta lisa (Figuras 1A e 1B). Tente não deixar pedaços grandes, já que não será dividida prontamente pela solução enzimática. </li><li> Transferir músculos picados para um tubo Falcon de 50 mL, e adicionam-se 5 ml de solução de colagenase (0,2% de colagenase do Tipo 2 em 10% de FBS em DMEM). Incubar a 37 ° C durante 60 min. </li><li> Triturar (para cima e para baixo com uma agulha 18 G) para homogeneizar a mistura (Figura 1C). Em seguida incubar ainda mais a mistura a 37 ° C durante 15 min. </li><li> Triturar novamente para homogeneizar mistura para dissociar-se em suspensão única célula. Adicionar 2% de FBS em DMEM até 50 ml para a suspensão de células individuais e misturábem. </li><li> Coloque um filtro celular (70 mM) em um tubo Falcon de 50 ml (Figura 1D). Transferir o sobrenadante contendo as células dissociadas em um coador de células, e a suspensão celular pipetar para cima e para baixo no filtro até que ela passe através. </li><li> Contar o número de células em hemocitômetro. Centrifugar os tubos a 2000 rpm a 4 ° C durante 5 min; aspirado e descartar o sobrenadante. </li><li> Ressuspender com 10 ml de 2% de FBS em DMEM. Centrifugar os tubos a 2000 rpm a 4 ° C durante 5 min; aspirado e descartar o sobrenadante. </li><li> Ressuspender com 200 mL de 2% FBS em DMEM e suspensão de células transferência para tubos de 1,5 ml de microcentrífuga. Normalmente, cerca de 2 x 10 6 células devem ser colhidas a partir de músculos de um rato. As células vão ser diluídas para uma concentração de 1 x 10 6 células em 100 ul de 2% de FBS em meio DMEM. </li></ol> 2. Anticorpo Coloração e separação com MACS Durante o seguinte procedures, manter condições estéreis utilizando tampões estéreis. Cada volume de anticorpos adicionados e meio de suspensão de célula é calculada para as células dos músculos inteiros de um rato. Se as células são colhidas a partir de duas ou mais ratos, a quantidade de reagentes deve ser optimizado. <ol><li> Adicionar 1 ml de cada um CD31-PE, CD45-PE, Sca-1-PE, e Integrina anticorpo α7 em 200 ul de suspensão de células. Incubar em gelo durante 30 min. </li><li> Lavar as células: Após a incubação, adicionar 1 ml de 2% de FBS em DMEM na suspensão de células no tubo de 1,5 ml e centrifugar a 2000 rpm a 4 ° C durante 3 min. Repita esta etapa duas vezes. </li><li> Aspirar e desprezar o sobrenadante. </li><li> Ressuspender as células com 200 mL de 2% FBS em DMEM, e adicionar 10 ml de Anti-PE esferas magnéticas. Incubar em gelo durante 30 min. </li><li> Lavar as células: Após a incubação, adicionar 1 ml de tampão de MACS para a suspensão de células no tubo de microcentrífuga de 1,5 ml, e, em seguida, centrifugar a 2000 rpm a 4 ° C durante 3 min. Repita este passo twgelo. Note-se que as células deverão ser lavadas pelo tampão MACS antes de ser separada por coluna magnética. </li><li> Aspirar e desprezar o sobrenadante. Ressuspender as células com 1,0 ml de tampão de MACS. </li><li> Configure coluna LD em uma placa magnética, e lavar a coluna com 2,0 ml de tampão MACS (Figura 1E). </li><li> Transferência da suspensão de células para dentro da coluna de LD, e recolher a fracção de escoamento num tubo de 1,5 ml. Esta fracção contém células PE-negativo. </li><li> Centrifugar a 2000 rpm a 4 ° C durante 3 minutos, aspirado e desprezar o sobrenadante. </li><li> Suspenda as células com 200 ul de 2% de FBS em DMEM, e adicionar 10 ul de IgG anti-ratinho esferas magnéticas. Incubar em gelo durante 30 min. </li><li> Lavar as células: Após a incubação, adicionar 1 ml de tampão de MACS para a suspensão de células em 1,5 ml de tubo e, em seguida, centrifugar a 2000 rpm a 4 ° C durante 3 min. Aspirar e desprezar o sobrenadante. Repita esta etapa duas vezes. Ressuspender as células com 500 ul de tampão de MACS. </li><li> Configure coluna MS em uma placa magnética, e lavar a coluna com 500 mL de tampão MACS (Figura 1F). </li><li> Transferência suspensa solução de células na coluna MS, e descartar a fração de fluxo (células α7 negativo integrinas). </li><li> Enxágüe com 1 ml de tampão MACS, e repita esta etapa duas vezes. </li><li> Depois de enxaguar, remover a coluna a partir do campo magnético do separador. Aplicar 1,0 ml de tampão de MACS para a coluna, e eluir as células magneticamente marcadas (células α7-positivo Integrina) para um tubo de microcentrífuga de 1,5 ml, empurrando o êmbolo da seringa a partir do topo da coluna. Recolhe-se o fluxo de passagem para um tubo de 1,5 ml. Repete-se a eluição com 1,5 ml de tampão de MACS e recolher o fluxo de passagem. </li><li> Centrifugar a 2000 rpm a 4 ° C durante 3 minutos, aspirado e desprezar o sobrenadante. </li><li> Ressuspender as células purificadas com 1 ml de meio de mioblasto (FBS a 20% continha Hams F-10 com bFGF), e células da placa em Matrigel-revestidas 10 centímetrosplaca com 8 ml de mioblasto Médio (5 ml em seis centímetros placa) (Figura 1G). Note-se que de 1-2 x 10 5 células pode, potencialmente, ser isolado a partir de um músculo intacto do rato. </li></ol> 3. Manutenção <ol><li> Alimentar as células a cada dois dias com meio mioblasto. O aparecimento de mioblastos em crescimento é de uma pequena e rodada expressando MyoD (Figura 2) e Pax7 (dados não mostrados) no seu núcleo. </li><li> Mioblastos devem ser passadas antes dos 50% de confluência ou quando iniciar a fusão celular. Depois de enxaguar uma vez com PBS, incubar as células com uma solução de tripsina a 0,25% a 37 ° C durante 3 minutos em uma incubadora de CO 2 e recolher células dissociadas com meio mioblasto. Após centrifugar células (1.000 rpm por 5 min), suspender com meio mioblasto e replate células em novas placas Matrigel-revestidos. Placas revestidas com colagénio pode ser usado após a passagem 3. Uma placa pode ser geralmente dividida em 3-5 placas. </li></ol> 4. Diferentiation <ol><li> Alimente novamente com Diferenciação Médio em dias alternados. </li><li> Por volta do dia 1 de Diferenciação Médio, mioblastos sair do ciclo celular e sofrem diferenciação em cadeia pesada da miosina (MHC) positivo miócitos. Estes myoctyes começar a fusão das células com o outro para gerar miotubos multinucleados. Tipicamente, a maioria dos mioblastos tornar miócitos MHC-positivos mononucleares de diferenciação ou miotubos (Figura 2) por dia 3-5 na Diferenciação Médio. </li></ol> 5. Mioblasto Transplante em mouse para Skeletal regeneração muscular <ol><li> Anestesiar o rato com Avertin (250 mg / kg) por via intraperitoneal (IP) de injecção. </li><li> Raspar o cabelo da pele em torno do músculo tibial anterior (TA). Vinte e quatro horas antes do transplante de mioblastos, 10 uM CTX (50 ul) é injectada por via intramuscular para o Node / músculo de ratinho SCID TA para induzir a regeneração do músculo através de uma seringa de 31 L de insulina através da pele raspada (Figura60, 3). </li><li> Mioblastos em proliferação são dissociados com uma solução de tripsina a 0,25% e centrifugadas a 1.000 rpm durante 5 min. Aspirar e desprezar o sobrenadante. Ressuspender 1 x 10 6 células, com 50 ul de 2% de FBS em DMEM. Transferir as células em suspensão com uma seringa de insulina de 31 L. </li><li> Os ratinhos receptores serão anestesiados com Avertin (250 mg / kg) por injecção IP, e os 1 x 10 6 mioblastos (Figura 2) são injectadas por via intramuscular em regeneração muscular TA. </li><li> Colheita TA muscular por 1-4 semanas após a injecção das células para análise histológica (Figura 3). </li></ol>

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Neste protocolo, as células satélites quiescentes podem ser facilmente purificadas a partir de músculo esquelético de ratinhos adultos por digestão com colagenase e de separação MACS mediada por anticorpos da superfície. Este método leva cerca de 6 horas e não precisa de nenhum equipamento caro, como uma máquina de FACS. Além disso, este método é relativamente barato em comparação com a superfície mediada por anticorpo de separação FACS. Uma maior produção de células quiescentes de satélite també...

Células satélites musculares são uma pequena população de células-tronco miogênicas localizadas abaixo da lâmina basal das fibras musculares esqueléticas. Eles são caracterizados pela expressão de Pax7, Pax3, c-Met, M-caderina, CD34, Syndecan-3, e calcitonina de 1-3. Células satélites têm provado ser responsável para a regeneração muscular, as células-tronco musculares. No músculo adulto, as células satélites são normalmente mitose repouso 4-8. Após lesão, as células satélites são ativadas, iniciar expressão de MyoD e entrar no ciclo celular para expandir a sua descendência, chamadas de células precursoras miogênicas ou mioblastos 3. Depois de vários ciclos de divisão celular, mioblastos sair do ciclo celular e fusível para o outro, a fim de sofrerem diferenciação em miotubos multi-nucleadas, seguido por fibras musculares maduras. Mioblastos isolados de músculo adulto pode facilmente ser expandidas ex vivo. A capacidade para mioblastos para tornar as fibras musculares na regeneração muscular eformar fibras musculares ectópica em tecidos nonmuscle é explorada por transplante de mioblastos, uma aproximação terapêutica potencial para a distrofia muscular de Duchenne (DMD), 4, disfunção urológica 9, e a insuficiência cardíaca 10. Com efeito, os mioblastos foram transplantadas com sucesso no músculo de ambos mdx (modelo DMD) e ratos doentes DMD 11-14. Os mioblastos normais injectados fundir com as fibras musculares de acolhimento para melhorar a histologia e função do músculo doente. Trabalhos anteriores demonstraram que subpopulações de mioblastos são mais células-tronco semelhantes e permanecem em um estado indiferenciado mais no músculo durante a regeneração muscular 5. Trabalhos recentes mostraram que as células satélites recém-isoladas de músculo adulto conter uma população de células-tronco como que exibe o enxerto mais eficiente e atividade auto-renovação na regeneração muscular 5-8. Portanto, a purificação de uma população pura de células satélites quiescentes de mu esquelético adultoesclerose é essencial para compreender a biologia de células satélites, mioblastos e regeneração muscular, e para o desenvolvimento de terapias baseadas em células. No entanto, os métodos de purificação de correntes de potenciais células satélites quiescentes requerem a utilização de um activado por fluorescência celular separação caro (FACS) da máquina 1,2,6-8. Além disso, a exposição ao laser FACS tende a induzir a morte celular durante a separação, o que faz com que o rendimento mais baixo de células quiescentes de satélite 15. Aqui, apresentamos um novo método para a purificação rápida, econômica e confiável de células satélites quiescentes de rato adulto músculo esquelético. Este método utiliza dissociação enzimática seguido de célula magnética ativado triagem (MACS). A seguir ao isolamento de células satélites quiescentes puros, estas células podem ser cultivadas para obter um grande número de mioblastos após várias passagens. Mostramos também que a injeção intramuscular dessas células satélites quiescentes recém-isoladas ou ex vimioblastos vo expandidos podem ser transplantadas para cardiotoxina (CTX) induzida regeneração do músculo esquelético do rato para examinar a contribuição de células derivadas de doadores para regenerar as fibras musculares, bem como para os compartimentos de células satélites para o exame de atividades de auto-renovação.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Collagenase Type 2</td>
			<td>Worthington</td>
			<td>CLS-2</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Marigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Invitrogen</td>
			<td>10569010</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Collagen (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td>100 mg (3-4 mg/ml)</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099-500ML</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>bFGF, human, Recombinant</td>
			<td>Gibco-Invitrogen</td>
			<td>PHG0263</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A5611-1G</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>Ham&#8217;s F10 Medium</td>
			<td>Gibco-Invitrogen</td>
			<td>11550-043</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>3600511</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco-Invitrogen</td>
			<td>26050088</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Penicillin/Streptmycin</td>
			<td>Gibco-Invitrogen</td>
			<td>15640055</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco-Invitrogen</td>
			<td>14190144</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Gibco-Invitrogen</td>
			<td>25200072</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>18G needle with 12cc Syringe</td>
			<td>Fisher Scientific</td>
			<td>22-256-563</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#956;m)</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml tube</td>
			<td>BD Biosciences</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 ml tube</td>
			<td>BD Biosciences</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm tissue culture plate</td>
			<td>BD Biosciences</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10 ml disposable pipet</td>
			<td>BD Biosciences</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody-PE</td>
			<td>eBiosciences</td>
			<td>12-0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD45 antibody-PE</td>
			<td>eBiosciences</td>
			<td>30-F11</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Sca1 antibody-PE</td>
			<td>eBiosciences</td>
			<td>Dec-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;7 antibody</td>
			<td>MBL International</td>
			<td>ABIN487462</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini &#38; MidiMACS Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-632</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>LD Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Sigma Aldrich</td>
			<td>C9759-1MG</td>
			<td>Stock 10 &#956;M in PBS</td>
		</tr>
		<tr>
			<td>31G Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>328438</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Microcentrifuge (Microfuge 22R)</td>
			<td>Beckman Coulter</td>
			<td>368826</td>
			<td></td>
		</tr>
		<tr>
			<td>S241.5 Swinging Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>368882</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge (Allegra X-22R)</td>
			<td>Beckman Coulter</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr>
			<td>Nod/Scid immunodeficient mice</td>
			<td>Charles River Laboratories</td>
			<td>Strain Code 394</td>
			<td>Use 2 months old mice</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Name of the reagent</td>
			<td></td>
			<td></td>
			<td>Recipie</td>
		</tr>
		<tr>
			<td>10% and 2% FBS DMEM</td>
			<td></td>
			<td></td>
			<td>DMEM (Gibco-Invitrogen #10569010) with 10% or 2% FBS (Fisher Scientific #03600511) and 1% Penicillin/Streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>0.2% Collagenase solution</td>
			<td></td>
			<td></td>
			<td>Collagenase Type 2 (Worthington, #CLS-2), Stock: 50 ml: 100 mg Collagenase Type 2 in 10% FBS DMEM.</td>
		</tr>
		<tr>
			<td>10% Matrigel solution</td>
			<td></td>
			<td></td>
			<td>Matrigel (BD Biosciences: #356234) is placed on ice for thawing overnight. Five ml Matrigel is dilute by 45 ml DMEM and 5 ml aliquots are stored at -20&#176;C until use.</td>
		</tr>
		<tr>
			<td>Matrigel-coated plate</td>
			<td></td>
			<td></td>
			<td>Five ml of 10% Matrigel solution is placed on ice for thawing and is used for coating 10 cm plate at room temprature for 1 minutes. The plate is placed in 5% CO2 incubator at 37&#176;C for 30 minute after removing Matrigel solution, and let the plate dry in culture hood for another 30 minutes. Removed 10% Matrigel solution is stored at -20&#176;C for reusing.</td>
		</tr>
		<tr>
			<td>0.01% Collagen solution</td>
			<td></td>
			<td></td>
			<td>Mix to final: 0.01% Collagen (Collagen, Rat Tail: BD Biosciences #354236) in 0.2% acetic acid (320099-500ML) in ddH2O.</td>
		</tr>
		<tr>
			<td>Collagen-coated plate</td>
			<td></td>
			<td></td>
			<td>Add 5 ml or 2 ml of Collagen solution to a 10 cm or 6 cm tissue culture plate and let sit at room temperature for three hours. Then, aspirate off liquid and allow to dry in culture hood for 30 min to overnight. Plates can be stored at room temperature for several months.</td>
		</tr>
		<tr>
			<td>bFGF stock solution</td>
			<td></td>
			<td></td>
			<td>bFGF, Human, Recombinant (Gibco-Invitrogen #PHG0263, 1 mg) is dissolved with 0.1% BSA solution consisting of 1 mg BSA (Sigma-Aldrich #A5611-1G) and 2 ml ddH2O (0.5 mg/ml bFGF). Aliquot 20 &#956;l in 500 &#956;l microcentrifuge tubes and kept in -80&#176;C.</td>
		</tr>
		<tr>
			<td>Myoblast medium</td>
			<td></td>
			<td></td>
			<td>500 mL HAM&#8217;S F10 Medium (Gibco-Invitrogen #11550-043) supplemented with 20% FBS (Fisher Scientific #03600511), Penicillin/streptomycin (Gibco-Invitrogen #15640055), and 10 &#956;g of bFGF (20 &#956;l of bFGF stock).</td>
		</tr>
		<tr>
			<td>Differentiation medium</td>
			<td></td>
			<td></td>
			<td>500 mL DMEM (Gibco-Invitrogen #10569010) supplemented with 5% Horse serum (Gibco-Invitrogen #26050088) and 1% Penicillin/streptomycin (Gibco-Invitrogen #15640055).</td>
		</tr>
		<tr>
			<td>10 &#956;M Cardiotoxin stock</td>
			<td></td>
			<td></td>
			<td>1 mg Cardiotoxin (EMD Millipore #217504-1MG) is dissolved with 13.9 ml PBS.</td>
		</tr>
	</tbody>
</table>

isolation, culture, and transplantation of muscle satellite cells

Células satélites musculares são uma população de células-tronco necessárias para o desenvolvimento muscular esquelética pós-natal e da regeneração, sendo responsável por 2-5% dos núcleos sublaminal nas fibras musculares. No músculo adulto, as células satélites são normalmente mitose repouso. Após lesão, no entanto, as células satélites iniciar a proliferação celular para produzir os mioblastos, as suas progênies, para mediar a regeneração dos músculos. O transplante de mioblastos derivados de células-satélite tem sido amplamente estudada como uma possível terapia para várias doenças regenerativas incluindo distrofia muscular, insuficiência cardíaca e disfunção urológica. O transplante de mioblastos em Latossolo músculo esquelético, coração infartado, e disfuncionamento ductos urinários mostrou que mioblastos enxertados podem se diferenciar em fibras musculares nos tecidos do hospedeiro e apresentar melhora funcional parcial nestas doenças. Portanto, o desenvolvimento de métodos de purificação eficiente de células satélites quiescentes de muscl esqueléticoe, assim como o estabelecimento de culturas de células derivadas de mioblastos de satélite e métodos de transplante de mioblastos, são essenciais para a compreensão dos mecanismos moleculares por trás de células auto-renovação satélite, ativação e diferenciação. Além disso, o desenvolvimento de terapias à base de células para a distrofia muscular e outras doenças regenerativos são também dependentes desses factores. No entanto, os métodos de purificação de correntes de potenciais células satélites quiescentes requerem o uso de caras activada por fluorescência (FACS) de células máquinas. Aqui, apresentamos um novo método para a purificação rápida, econômica e confiável de células satélites quiescentes de rato adulto músculo esquelético por dissociação enzimática seguido de célula magnética ativado triagem (MACS). A seguir ao isolamento de células satélites quiescentes puros, estas células podem ser cultivadas para obter um grande número de mioblastos após várias passagens. Estes recém-isoladascélulas satélites quiescentes ou ex vivo mioblastos expandidas podem ser transplantadas em cardiotoxina (CTX) induzida por rato em regeneração do músculo esquelético para examinar a contribuição de células derivadas do dador para a regeneração das fibras musculares, bem como para compartimentos celulares por satélite para o exame da auto-renovação actividades.

Células satélites quiescentes recentemente isoladas exibir uma forma pequena e redonda (Figura 1G) e Pax7 expressa como um marcador definitivo para as células satélites quiescentes. Mais de 90% das células recentemente isoladas expressam Pax7 (Figura 1H e 1I). A maioria das células contaminadas são a partir de células de sangue que não crescem de forma eficiente in vitro seguintes condições de cultura de mioblastos. Assim, mioblastos derivados de cé...

Watch this Scientific Journal Video about Isolation, Culture, and Transplantation of Muscle Satellite Cells at JoVE.com

Isolation, Culture, and Transplantation of Muscle Satellite Cells

O isolamento e cultura de uma população pura de células satélites quiescentes, uma população de células estaminais musculares, é essencial para a compreensão da haste músculo biologia celular e regeneração, bem como o transplante de células estaminais para terapias em distrofia muscular e outras doenças degenerativas.

Isolamento, cultura e transplante de células musculares satélite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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Fibroblast Transformation and Muscle Stem Cells

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41.0K visualizações.  University of Minnesota Medical School.  O isolamento e cultura de uma população pura de células satélites quiescentes, uma população de células estaminais musculares, é essencial para a compreensão da haste músculo biologia celular e regeneração, bem como o transplante de células estaminais para terapias em distrofia muscular e outras doenças degenerativas. 

Vídeo: Isolation, Culture, and Transplantation of Muscle Satellite Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolamento e Transplante de Células-Tronco Hematopoéticas (HSCs)

Biologia

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

Isolamento da Human Umbilical células musculares arteriais (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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 and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

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 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 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 and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e Cultura de miofibras individuais e suas células satélites do músculo esquelético

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Isolamento e Cultura do mouse primárias células pancreáticas acinares

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolamento de artéria pulmonar células musculares lisas de camundongos recém-nascidos

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...