Este trabalho foi financiado pelos Institutos Nacionais de Saúde (R01HL056046 para PAL e K99HL116769 para AJT) e do Instituto de Pesquisa do Hospital Infantil Nationwide (para PAL e AJT).

Declaração de Ética: Este estudo foi realizado de acordo com os Institutos Nacionais de Diretrizes de Saúde, e foi aprovada pelo Animal Care Instituição e do Comitê Use no Hospital Infantil Nationwide. 1. Preparação / Configuração Nota: Esta técnica de isolamento requer duas bobinas de aquecimento de Langendorff posicionados lado-a-lado sobre um suporte anelar, e ligado em paralelo a um banho de água circulante. <ol><li> Ligue banho de água circulante e ajustar a temperatura para alcançar uma temperatura final de 33-34 ° C na ponta da cânula de Langendorff (o banho de água utilizado nesta configuração é definido como 40 ° C devido a uma perda de 6-7 ° C em temperatura entre os tubos). Ligar as bombas de perfusão e definir a taxa de perfusão a 0,5 ml / min. <ol><li> Limpar ambas as bobinas de aquecimento com camisa de água, ligado tubagem 25 e de uma cânula de calibre, através de lavagem, cada um com 50 ml de etanol a 70%, seguido de 100 ml de água ultrapura esterilizada. </li><li> Limpar as bobinas de aquecimentoempurrando o ar com uma seringa e lava-se com 10 ml de Solução Salina Equilibrada de Hank (HBSS) sem vermelho de fenol. Deixar HBSS nas serpentinas de aquecimento e os tubos. </li><li> Anexar uma seringa de 10 ml estéril luer preenchido com temperatura ambiente de HBSS sem vermelho de fenol para o tubo. Certifique-se que não há bolhas de ar presas no interior dos tubos e aquecimento bobinas ao anexar a seringa HBSS-cheia, pois pode provocar embolias de ar durante a digestão para impactar negativamente o isolamento VSMC. </li></ol></li><li> Remover a cânula de calibre 25 a partir das bobinas de aquecimento e em outro lugar estéril seringa de 10 ml cheia com HBSS arrefecido em gelo, sem vermelho de fenol. Tenha isso em gelo até estar pronto para usar. Repita para o outro cânula. </li><li> Preparar solução de digestão enzimática. <ol><li> Pesar colagenase tipo 2 de acordo com o número do lote específico de 75 ml de solução de digestão para atingir uma concentração final de 300 Unidades / ml. </li><li> Dissolve-se a colagenase em 75 ml de HBSS com vermelho de fenol. Em seguida, adicione 1,5 ml de 00,1 mg / mL de inibidor de tripsina de soja e 75 ul de 1 M de CaCl2 para a mistura. </li></ol></li><li> Se utilizar a solução de digestão imediatamente, aquecê-la a 37 ° C. Se não, coloque-o a 4 ° C durante até 6 h. Esta solução deve ser feita diariamente. </li><li> Prepare a mídia de solução de paragem / chapeamento. <ol><li> Adicionam-se 100 ml de FBS inactivado pelo calor, 5 mL de L-glutamina, 5 ml de aminoácidos não essenciais, 5 ml de tampão HEPES, e 1 ml de primocin a 500 ml de alto teor de glucose (4,5 g / L) de DMEM. esterilização por filtração é recomendada. </li><li> Aliquota 6 ml da solução de paragem em cada um de doze tubos de 15 mL cónico, numerados de A1-A6, B1-B6 e, e colocar a 4 ° C. Alíquota de 50 ml de solução de paragem em uma cónico de 50 ml e em lugar 37 ° C incubadora. Nota: Este passo também pode ser preparado no dia antes do isolamento, se necessário. Esta solução de paragem irão ser utilizados como os meios de revestimento seguintes isolamento de células. </li></ol></li><li> Prepare duas seringas de 1 ml equipada com uma agulha de 30 Gcom 100 ul de heparina (1000 unidades / ml). </li><li> Corte um pedaço de 5 cm de 5-0 seda cirúrgico. Dê um nó overhand solto na seda cirúrgico com uma extremidade duas vezes, enquanto a outra. Coloque sobre a cânula de calibre 25, mas não aperte. Repita para o outro 25 G cânula. </li><li> Encher 35 mm quatro placas de Petri estéreis (# 1-4) com 2 ml de solução estéril, arrefecida com gelo, de HBSS sem vermelho de fenol. Manter em gelo. </li></ol> 2. A eutanásia, Isolamento Coração e Canulação <ol><li> Antes de isolamento coração, garantir que a solução de digestão e 50 ml cónico de solução de paragem são quentes. Retire a seringa HBSS-preenchido com anexado 25 G cânula do gelo e coloque em uma bureta grampo (ou braçadeira de suporte de anel) em um suporte de anel. </li><li> Anestesiar ratos usando 3% isoflurano. Confirmar o mouse é anestesiado por um toque do dedo do pé em cada pata traseira. Se as contrações musculares das pernas, o mouse ainda não está anestesiado então espere 1 minuto e repita o toque do dedo do pé. </li><li> Expor a veia jugular através da remoção do pêlo de umND pele na face anterior do pescoço com uma tesoura. Em seguida, utilize uma pinça para remover cuidadosamente qualquer gordura ao redor da veia jugular. Após a remoção de gordura, injectar-se lentamente 100 ul de heparina (1000 unidades / ml). Coloque uma esponja cirúrgica sobre a veia para evitar sangramento excessivo. </li><li> Após 1 min, de peito aberto para expor coração, coração elevador com uma pinça curva e extirpar-lo com uma tesoura, certificando-se de manter a aorta ascendente intacta através do primeiro tronco braquiocefálico. Colocar imediatamente o coração em um prato de 35 mm (# 1) com HBSS arrefecido em gelo, sem vermelho de fenol e enxaguar. Nota: Execute as seguintes 4 etapas usando um escopo de dissecação. </li><li> Posicionar a 25 L de uma cânula que é montado sobre o anel de suporte num ângulo de 45 °, de modo que a ponta está situada logo abaixo da superfície de HBSS arrefecido com gelo no prato # 2 e é claramente visível através do microscópio de dissecção. Transfira o coração para prato # 2. </li><li> Utilizando uma pinça Dumont, expor a aorta, removendo suavemente o excesso de tecido adiposo através de dissecção romba para prevent de perfuração ou ruptura da aorta. Em seguida, utilize uma pinça para deslizar a aorta sobre o G cânula 25. </li><li> Orientar a cânula para baixo através da aorta, perto, mas não além da válvula aórtica, o que irá comprometer a integridade da válvula e, portanto, a perfusão retrógrada. Uma vez que a cânula está no lugar, deslize a seda pré-amarrado sobre a aorta, e aperte. Amarrar um segundo nó para formar um nó quadrado em torno da aorta para proteger o coração. </li><li> Uma vez que a aorta está firmemente amarrado, delicadamente lavar o sangue restante da circulação coronária com o HBSS gelada na seringa anexado ao G cânula 25. Tome cuidado para não apertar demais para evitar a pressão excessiva coronária. Apenas um flush muito lento e suave é necessária para completar o flush. o uso da aorta livre de vazamentos também pode ser observada durante este rubor suave. </li><li> Ligar a bomba de perfusão para iniciar o fluxo de HBSS. Remover a 25 L de uma cânula (com o coração em anexo) a partir da seringa HBSS-cheia, tendo o cuidado demanter o hub cânula preenchido com HBSS, e conecte-o a quente bobinas de aquecimento HBSS-cheia. Comece a perfusão do coração com a bomba de perfusão com um caudal de 0,5 mL / min durante 8 minutos. </li><li> Após o primeiro coração é canulada e está a ser perfundido no sistema, repita os passos de 2,1-2,8 para o segundo coração. Estes isolamentos serão escalonadas, para cada coração, portanto, planejamento e cronograma cuidadoso são necessários. </li></ol> 3. Digestão <ol><li> Após a lavagem com HBSS, alterar a seringa de 10 ml de HBSS actualmente na bomba para uma cheia com 10 ml de solução de digestão de pré-aquecido. Perfundir cada coração com solução de digestão durante 12 min a um caudal de 0,5 mL / min. Não coletar frações decorrentes dessa primeira digestão, pois isso irá conter todo o sangue remanescente e células endoteliais. </li><li> Após 12 min, alterar a taxa de perfusão a 0,4 ml / min, colocar uma cónico de 15 ml (tubo de A1 ou B1, correspondente ao primeiro ou segundo coração para cada digestão) preenchido com solut paragem frioion em um balde de gelo sob o coração, a fim de iniciar a recolha de perfusato enriquecido em VSMC. </li><li> Após 15 min, alterar a recolha cónica para A2 ou B2 e tubo de centrifugação cónico tubo A1 e B1 a 89 xg durante 10 min imediatamente após a recolha de ambas as fracções. Quando a seringa cheia com a solução de digestão na bomba tem menos do que 1 ml de solução, substitua com um recém-cheia seringa de 10 ml da solução de digestão. </li><li> Após centrifugação, Aspirar o sobrenadante de cada tubo de recolha, deixando cerca de 0,5 ml. Ressuspender o sedimento de A1 com 2 mL de solução de paragem quente (ou meios plating). Adicionar a ressuspensão de tubo de A1 para o tubo B1 e tubo lugar no 37 ° C incubadora com a tampa solta. </li><li> Repita os passos 3.3 e 3.4 até que todos os tubos (por meio de A6 e B6) são utilizadas para um tempo total de recolha de 90 min. A 80 min de digestão, cortar abrir o ápice coração para expulsar as células que possam estar presas dentro do ventrículo e recolher essas células em A6 tubo e B6, respectively. Nota: Ocasionalmente, o coração irão cair da cânula antes do ponto de tempo de 90 min. Se isto ocorrer, recuperar o coração e o local em uma estéril 35 milímetros prato de Petri cheio com 2 ml de solução de digestão. Cortar o vértice do coração e lave o coração com solução de digestão. Em seguida, filtra-se os 2 ml de solução de digestão através de um filtro de 100 uM de células estéril para um cónico de 50 ml e adicionar ao tubo A6 ou B6. </li><li> Após cada rodada de tubos subsequentes (A2 e B2), adicione as VSMC ressuspensos às tubo de coleta anterior (combinado A1 e B1). Além disso, tome cuidado para não deixar a solução de digestão na bomba de correr para fora e mudar os 10 ml seringas conforme necessário durante toda a digestão. </li><li> Depois de todos os tubos são combinados, centrifugar a suspensão rica em CMLV a 89 xg durante 10 min. </li><li> Aspirar tanto sobrenadante dos tubos de colheita quanto possível. Ressuspender o sedimento remanescente em 2 ml de meios de comunicação e plaqueamento na placa por um prato de cultura de 35 mm. Lugar cultura prato iN incubadora C 37 ° durante 24 h. </li></ol> Cultura 4. celular Nota: Conclua as etapas restantes no gabinete de biossegurança. <ol><li> Após 24 horas, aspirar cuidadosamente a mídia chapeamento. A cultura terá normalmente uma grande quantidade de detritos. Para lavar as células, adicionar 2 ml de quente, PBS estéril e bata suavemente o prato contra dois dedos durante a rotação no sentido horário. </li><li> Depois de rodar 360 °, aspirar o PBS e repetir uma segunda vez. Aspirar o PBS e adicionar outro 2 ml de estéril, PBS quente. Verifique a placa de cultura sob o microscópio para se certificar de que há pouca ou nenhuma restos no prato. confluência celular também pode ser determinada neste momento. </li><li> Se os restos permanece na placa de cultura, repita o passo 4.2. Se a placa de cultura é livre de detritos, aspirar o PBS e adicionar 2 ml de mídia chapeamento quente. </li><li> Após 24 h, lavar as células novamente com quente, PBS estéril para remover quaisquer detritos final ou células mortas da cultura. Aspirar o PBS e pate mais 2 ml de meio de plaqueamento quente sobre as células. </li><li> Verifique sobre as células a cada dia e alimentar a cada 48 horas com a mídia chapeamento aquecido frescos. Neste ponto, as células podem ser utilizadas para ensaios de cultura de células padrão. As VSMC deve ser de aproximadamente 80% confluentes 4-5 dias após o plaqueamento, embora possa levar até 7 dias, até que o processo é aperfeiçoado. Se a divisão de passagens subsequentes, utilizar uma quantidade máxima de 1: 4 35 mm pratos (ou a uma área comparável de placas de cultura). </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

O objetivo deste estudo foi adaptar protocolos de isolamento de células existentes para aumentar o rendimento de vascular coronariana suave de corações de ratinho. A maior parte do trabalho pioneiro na biologia do músculo liso vascular foi realizada com células de músculo liso de aorta de rato cultivadas. Estes estudos deram conhecimento fundamental dos mecanismos moleculares que controlam o crescimento VSMC, migração e hipertrofia 7. No entanto, como o campo progrediu, tornou-se evidente que VSMC fen...

O objectivo deste método é isolar as células musculares lisas vasculares (VSMCs) a partir da circulação coronária murino para uso em cultura de células e ensaios de cultura de células padrão. Nós desenvolvemos esta técnica para avaliar os mecanismos moleculares da remodelação vascular na diabetes. Temos relatado anteriormente para dentro remodelação hipertrófica nas arteríolas coronárias septais no modelo de camundongo db / db de diabetes 1. Devido à quantidade limitada de tecido encontrado nas coronárias septais de murino, as técnicas experimentais padrão investigaram as alterações da proteína (por exemplo, transferência de Western) em ratinhos db / db e de controlo são difíceis na melhor das hipóteses. Além disso, temos mostrado previamente que o bloqueador do receptor de angiotensina (BRA) losartan reduz a remodelação observada em ratinhos db / db 2. Por conseguinte, o isolamento de VSMCs de primários a partir da circulação coronária nos permite investigar mudanças em CMLV fenótipo ou vias de sinalização activadas em ratos diabéticos, que podem ser contriting para adversa remodelação das arteríolas coronárias. Numerosos estudos têm elucidado vias de sinalização que utilizam canónicos VSMCs isoladas a partir da aorta de roedores, em vez de em cada leito vascular específico. No entanto, nós demonstramos vascular-cama remodelação específica em coronárias, aorta, e circulações mesentéricas de ratinhos db / db 1, sugerindo que os VSMCs em cada leito vascular pode ser diferente. Por conseguinte, é necessário isolar a partir de VSMCs de cada leito vascular, a fim de melhor compreender as alterações patológicas que ocorrem em cada conjunto de VSMC. Há uma infinidade de diferentes métodos para o isolamento e cultura de VSMCs de aorta. No entanto, actualmente, há apenas um estudo que foi publicada no isolamento de VSMCs de rato a circulação coronária 3. Teng et ai. foi o primeiro a relatar um método para o isolamento de VSMCs de rato a circulação coronária; no entanto, temos alterado o protocolo significativamente como eles também isolado cel endotelialls. Outros laboratórios também têm utilizado o protocolo de Teng et al. Isolar miócitos arteriais coronárias e das vias aéreas células musculares lisas 4,5. As alterações que tenham incorporado irá produzir uma população de células altamente enriquecidas em VSMC da circulação coronária. A perfusão retrógrada do coração dos mamíferos isolado, ou técnica de Langendorff, foi criada em 1897, 6 por Oscar Langendorff e ainda hoje é amplamente utilizado para o isolamento de células cardiovasculares. A técnica aqui apresentada, juntamente com o avanço das modificações genéticas modernas de murino, proporciona uma valiosa ferramenta para investigação mais detalhada do comportamento molecular de VSMC da circulação coronária.

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid (phenol red)</td>
			<td style="width:143px;">Life Technologies</td>
			<td>14170-161</td>
			<td></td>
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			<td style="width:143px;"></td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5.0 ml heating coil with degassing bubble trap</td>
			<td>Radnoti</td>
			<td>158830</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">11 plus pump</td>
			<td style="width:143px;">Harvard Apparatus</td>
			<td style="width:119px;">70-2208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Circulating heated water pump</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>any brand will work</td>
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	</tbody>
</table>

isolation of murine coronary vascular smooth muscle cells

Embora o isolamento e cultura de células de músculo liso vascular (VSMCs) a partir dos navios de grande porte está bem estabelecida, procurou-se isolar e cultivar as VSMCs da circulação coronária. Corações com arcos aórticos intactos foram removidos e perfundidos através de Langendorff com solução de digestão retrógrada contendo 300 unidades / mL de colagenase tipo II, 0,1 mg / mL de inibidor de tripsina de soja e 1 M de CaCl2. Os perfusatos foram recolhidas a intervalos de 15 minutos durante 90 minutos, sedimentadas por centrifugação, ressuspensas em meio de plaqueamento, plaqueadas e em placas de cultura de tecidos. As VSMC foram caracterizados pela presença de SM22α, α-SMA, e vimentina. Uma das principais vantagens da utilização desta técnica é a capacidade de isolar VSMCs da circulação coronária de ratos. Embora o pequeno número de células obtidas podem limitar algumas das aplicações para as quais as células podem ser utilizados, VSMCs coronárias isoladas podem ser utilizadas em uma variedade de técnicas de cultura de células bem estabelecidos e assays. Estudos investigando VSMC de camundongos geneticamente modificados pode fornecer mais informações sobre a estrutura-função e os processos de sinalização associados com patologias vasculares.

Devido ao aspecto inovador da nossa técnica de isolamento de VSMC coronária, procurou-se determinar a pureza do isolamento de células. As VSMC coronárias de rato foram identificados com base na sua morfologia e coloração de imunofluorescência até passagem 2. Com base na morfologia das células em cultura após a primeira lavagem, o procedimento de isolamento remove eficazmente os miócitos cardíacos e células endoteliais. As VSMCs de reter a sua morfologia até passagem 2 <stro...

Watch this Scientific Journal Video about Isolation of Murine Coronary Vascular Smooth Muscle Cells at JoVE.com

Isolation of Murine Coronary Vascular Smooth Muscle Cells

O objectivo deste protocolo é demonstrar as técnicas de isolamento e cultivo de células musculares murino primários liso vascular (VSMC) da circulação coronária. Uma vez que as VSMCs foram isolados, eles podem ser usados ​​para várias técnicas de cultura convencionais.

Isolamento de Murino vasculares coronários células musculares lisas

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

isolation-of-murine-coronary-vascular-smooth-muscle-cells

Research

JoVE Journal

Biology

14.0K visualizações.  The Ohio State University College of Medicine.  O objectivo deste protocolo é demonstrar as técnicas de isolamento e cultivo de células musculares murino primários liso vascular (VSMC) da circulação coronária. Uma vez que as VSMCs foram isolados, eles podem ser usados ​​para várias técnicas de cultura convencionais. 

Vídeo: Isolation of Murine Coronary Vascular Smooth Muscle Cells

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Focal Ca 2 + Detecção de transitórios no músculo liso

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

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 Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Isolamento e diferenciação de células estromais vasculares para Células Bege / Brite

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

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 ...

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 of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

O isolamento de células endoteliais de Murino Válvula

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

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 ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Isolamento de células de camundongo Coronária endoteliais

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Lesão por esmagamento aórtico murino: um eficiente<em&gt; In Vivo</em&gt; Modelo de proliferação de células musculares lisas e função endotelial

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...