Name: isolation and primary culture of mouse aortic endothelial cells
Uploaded: 2016-12-19T13:00:00.000+00:00
Duration: 8 min 20 s
Description: Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

This study is supported by American Heart Association Scientist Development Grant 13SDG16930098 and the National Science Foundation of China Youth Award 81300240 (PI: Wang). We thank Roberto Mendez from Wayne State University for assisting in the preparation of the manuscript.

1. Isolamento de aorta de ratinhos Todos os procedimentos aqui descritos foram aprovados pelo Comitê Cuidado e Uso Institucional animal de Wayne State University. <ol><li> Coloque o mouse na câmara de indução da máquina de anestesia. Defina o fluxo de isoflurano a 4% em conjunto com 25% de ar fresco e 75% de O 2. Anestesiar o rato por inalação de isoflurano (4% para indução, ± 1,0% para manutenção) em conjunto com 25% de ar fresco e 75% de O 2, por intermédio da câmara de indução seguida por um cone de nariz dedicado. NOTA: Anestesia com isoflurano pode ser entregue em 75% de ar, de oxigénio / 25% ou 100% em oxigénio. </li><li> Verifique o mouse a cada 5 minutos até que o nível adequado de anestesia é alcançado (falta de retirada pitada tep). </li><li> Aqui o rato para fora da câmara de indução e continuar a inalação de isoflurano que o cone do nariz dedicado que é ligado ao equipamento de anestesia. alternar aisoflurano fluir para 1% para manter a anestesia. </li><li> Embora seja sob anestesia, coloque o mouse sobre um bloco de cirurgia, posicionadas em suas costas. Use fita de laboratório para proteger os membros. </li><li> Use uma lâmpada de aquecimento para manter o rato quente. Mantenha a lâmpada em uma distância adequada do mouse para que o rato não vai mais ao calor. </li><li> Pulveriza-se a caixa com 70% de etanol. </li><li> Use tesouras de dissecação para abrir o abdómen da linha média, e expor a aorta abdominal. Abrir a cavidade torácica e expor o coração e os pulmões. </li><li> Corte a aorta abdominal no meio com uma tesoura de dissecção para liberar o sangue. </li><li> Encha uma seringa de 1 ml (com 25 g de agulhas) com 1 ml de PBS contendo 1.000 U / ml de heparina. Injectar PBS contendo 1.000 U / ml de heparina no ventrículo esquerdo e a aorta perfundir. Empurrar o coração e o pulmão com uma pinça com as grandes artérias para o lado direito dos ratinhos para expor a aorta torácica. </li><li> remover rapidamente da aorta torácica utilizando micro-diforceps ssection e colocá-lo em 1x PBS gelado (estéril), em seguida, transportar o recipiente em uma capa de fluxo de ar laminar. </li><li> Inserir uma seringa de 1 ml, equipado com 25 agulhas G em uma das extremidades da aorta, e lavar suavemente a aorta com PBS gelado para remover o sangue. </li><li> Use uma pinça micro-dissecção para remover o máximo do tecido adiposo em anexo e pequenos vasos laterais possível. </li><li> Imediatamente transferir a aorta para meio de crescimento endotelial. </li><li> Corte a aorta em rodelas de 1 mm usando uma lâmina de bisturi estéril. Colheita aproximadamente 8 - 10 anéis por aorta. </li><li> Abra cada anel aórtico usando um par de tesouras micro-dissecção. </li></ol> 2. As sementes dos segmentos da aorta em Matrix <ol><li> Quando não estiver em uso, mantenha a matriz reduzida do fator de crescimento em -20 ° C para evitar a solidificação. Coloque a matriz reduzida pelo factor de crescimento em 4 ° C, pelo menos durante a noite para permitir um descongelamento completo. </li><li> Pré-arref ecimento de 6 poços de placa e pontas de pipeta para -20 ° C durante pelo menos10 min. Colocar a placa de 6 poços em gelo e um revestimento das cavidades da placa com 1 ml de matriz sem introduzir quaisquer bolhas de ar. Colocar a placa num incubador de 37 ° C durante 20 min para permitir que a matriz se solidificar. </li><li> Implantar as peças da aorta para a matriz solidificada usando uma pinça micro-dissecção estéreis. Coloque as peças lúmen de cabeça para baixo sobre a matriz, sem tocar no endotélio. Colocar 3 - 4 segmentos aórticos próximos uns dos outros na matriz. </li><li> Adicionar meio de crescimento de células endoteliais apenas o suficiente para manter os segmentos molhado (~ 200 mL). </li><li> Incubar a placa a 37 ° C sob 5% de CO2 durante 4 a 6 h. No final do dia, adicionar meio apenas suficiente para cobrir os segmentos aórticos. </li><li> Observar o segmento da aorta sprouting periodicamente sob um microscópio de contraste de fase. Verifique o nível médio e crescimento celular a cada dia. Adicionar meio se necessário para manter segmentos da aorta cobertos. </li><li> No dia 4 th, remova gentilmente o meio e retire a segmentos da aorta from a matriz, utilizando uma agulha estéril sem interromper as células endoteliais que crescem. </li><li> Adicionar 2 ml de meio de crescimento de células endoteliais novo e permitir que as células endoteliais continuam a proliferar na matriz durante 2 - 3 dias. </li></ol> 3. Passaging inicial das células endoteliais da aorta do rato <ol><li> Revestimento de um novo frasco T12.5 com gelatina (0,1%), incuba-se a 37 ° C durante 30 min. </li><li> Lavam-se as placas cuidadosamente com matriz estéril 37 ° C de PBS. </li><li> Adicionar 2 ml de proteinase neutral (50 U / mL) para as placas de matriz e incubar à temperatura ambiente no balancim plataforma com agitação ocasional. Verifique as células em um microscópio de contraste de fase para garantir que a maioria das células são separadas. </li><li> Adicionar 2 mL de D-Val para inactivar a proteinase neutro. </li><li> Recolhe-se o sobrenadante cuidadosamente num tubo de centrífuga de 15 mL. Lava-se a placa com 2 ml de D-Val e recolher o sobrenadante. </li><li> Centrifugar a suspensão de células a 900 xg durante 5 min a RooTemperatura m. </li><li> Re-suspender o sedimento de células em 4 ml de meio de crescimento de células endoteliais e placa no balão T12.5 revestidos com gelatina. Incubar as células a 37 ° C, 5% de CO 2 durante 2 h. </li><li> Substituir o meio. Incubar as células a 37 ° C, 5% de CO2 até que eles são 85% - 90% confluentes. </li></ol> 4. Passaging das células de camundongo aórtica endoteliais <ol><li> Revestimento dois novos frascos T12.5 com gelatina (0,1%), incuba-se a 37 ° C durante 30 min. Pré-aqueça Tripsina-EDTA (tripsina a 0,25%, 0,02 EDTA em PBS) e PBS estéril a 37 ° C durante cerca de 15 min. </li><li> Lavam-se as células cuidadosamente com estéril 37 ° C de PBS. </li><li> Adicionar 0,5 mL de Tripsina-EDTA (tripsina a 0,25%, 0,02 EDTA em PBS) e incuba-se a 37 ° C durante ~ 1 min ou até que a maioria das células se virar. </li><li> Adicionar 2 ml de meio de crescimento de células endoteliais para parar a digestão. Pipetar a suspensão de células para cima e para baixo dentro dos frascos de várias vezes. Se necessário, utilizar uma célularaspador para recolher as células. </li><li> Recolha a suspensão celular num tubo de centrífuga de 15 mL. Centrifugar a suspensão de células a 900 xg durante 5 min à temperatura ambiente. </li><li> Re-suspender o sedimento de células em 4 ml de meio de crescimento de células endoteliais e da placa em balões de 2 T12.5 revestidas com gelatina. Incubar as células a 37 ° C, 5% de CO 2 durante 2 h. </li><li> Substituir o meio. Incubar as células a 37 ° C, 5% de CO2 até que eles são 85% - 90% confluentes. </li><li> Use as células endoteliais da aorta de rato após 2 - 3 passagens. </li></ol> 5. Caracterização das células de camundongo aórtica endoteliais <ol><li> Re-placa das células. <ol><li> Bata de uma placa de 6 poços com gelatina (0,1%), incuba-se a 37 ° C durante 30 min. Lavar a placa com PBS. </li><li> Pré-aqueça o Tripsina-EDTA e PBS estéril a 37 ° C durante cerca de 15 min. </li><li> Lavam-se as células com PBS estéril 3 vezes. Adicionar 0,5 mL de Tripsina / EDTA para as células, incubar durante ~ 1 min a 37 ° C óR até que a maioria das células se virar. </li><li> Adicionar 2 ml de meio de crescimento de células endoteliais para parar a digestão. Pipetar a suspensão de células para cima e para baixo dentro dos frascos de várias vezes. Se necessário, utilizar um raspador de células para recolher as células. </li><li> Recolha a suspensão celular num tubo de centrífuga de 15 mL. Centrifugar a suspensão de células a 900 xg durante 5 min à temperatura ambiente. </li><li> Descartar o sobrenadante, re-suspender as células em meio de crescimento de células endoteliais. Semear as células em placas de 6 poços revestidas com gelatina a uma densidade de 3 x 10 5 / poço. </li><li> Incubam-se as células durante a noite a 37 ° C, 5% de CO2 para permitir que as células se fixem à superfície da cultura. </li></ol></li><li> Dil-ac-LDL captado e Ulex -lectin vinculativo. <ol><li> Armazenar o 1,1&#39;-dioctadecil-3,3,3 &#39;, 3&#39;-tetramethylindo carbocianina marcado com perclorato de LDL acetilada (Dil-ac-LDL) a -20 ° C. Antes da coloração, descongelar Dil-ac-LDL em gelo. A solu estoque Dil-ac-LDLção é de 1 mg / mL. Armazenar a aglutinina marcado com FITC Ulex europaeus (Ulex -Lectin, 1 ug / mL) a 4 ° C. A solução Ulex -Lectin estoque é de 1 mg / mL. Loja Hoechst 33342 a 4 ° C. A 33342 solução de Hoechst é de 100 mcg / mL. </li><li> Adicionar 10 ul de solução de estoque Dil-ac-LDL a 1 mL de meio de cultura para obter uma concentração final de 10 ug / mL. </li><li> Antes da coloração, remover o meio velho e lavar as células em cada poço com o novo meio. </li><li> Retire o novo meio e adicione o meio de coloração Dil-ac-LDL. </li><li> Incubar as células a 37 ° C, 5% de CO 2 durante 1 h. </li><li> Lavam-se as células com PBS 1x estéreis 3 vezes. </li><li> Adicionar 1 ml de formol a 10% para fixar as células. Colocar a placa à temperatura ambiente durante cerca de 30 min. </li><li> Lavam-se as células com PBS 1x estéreis 3 vezes. Evite luz. </li><li> Adicione 1 mL estéril 1x PBS que contém 10 ml de Ulex -Lectin. </li><li> Incubar as células a temperatu quartore no escuro durante 1 h. </li><li> Remover o Ulex-LECTINA por lavagem das células com PBS 1x estéreis 3 vezes. </li><li> Adicionar a Hoechst 33342 para concentração final de 1 ug / ml, incubar 10 min no escuro. </li><li> Ver a fluorescência do Dil-ac-LDL (vermelho, comprimento de onda de excitação de 576 nm), Ulex -Lectin (verde, excitação comprimento de onda de 519 nm) e Hoechst (azul, excitação comprimento de onda de 361 nm) com um microscópio de fluorescência invertido. </li></ol></li><li> coloração fluorescente para CD31, VEGFR2, eNOS, VE-caderina e calponina. <ol><li> Loja anticorpo anti-ratinho CD31 conjugado com FITC, anticorpo eNOS, VE-caderina de anticorpo, anti-IgG de coelho conjugado com FITC, a 4 ° C no escuro. Loja anticorpo VEGFR2 em -20 ° C. </li><li> Lavam-se as células com PBS 1x estéreis 3 vezes. </li><li> Adicionar 1 ml de formol a 10% para fixar as células. Colocar a placa à temperatura ambiente durante cerca de 30 min. </li><li> Lavam-se as células com PBS 1x estéreis 3 vezes. </li><li> Para manchar CD31, adicione 1 mL de 1x estérilPBS que contém 10 ml de anticorpo CD31-FITC. Para manchar quer VEGFR2, eNOS, VE-caderina ou calponina, adicione 1 mL de estéril 1x PBS que contém 4 mL de VEGFR2, eNOS, VE-caderina ou calponina anticorpo primário, respectivamente. </li><li> Incubar as células em gelo durante 1 h no escuro. </li><li> Remover os anticorpos por lavagem das células com PBS estéril 3 vezes. </li><li> Para a coloração de VEGFR2, eNOS, VE-caderina ou calponina, adicionar 1 ml de PBS estéril 1x que contém 10 ul de IgG anti-coelho conjugado com FITC. Incubar as células em gelo durante 1 h no escuro. Remover o IgG por lavagem das células com PBS estéril 3 vezes. </li><li> Adicionar a Hoechst 33342 para concentração final de 1 ug / ml, incubar 10 min no escuro. </li><li> Ver a fluorescência de CD31, VEGFR2, eNOS, VE-caderina, calponina (verde, comprimento de onda de excitação de 518-535 nm) e Hoechst (azul, comprimento de onda de excitação de 361 nm) com um microscópio de fluorescência invertido. </li></ol></li></ol>

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The authors have nothing to disclose.

Este estudo demonstra um método simples para isolar e endotelial cultura células de uma aorta mouse sem qualquer equipamento especial. A coloração por imunofluorescência indicou que a maioria das células eram células endoteliais após a segunda passagem. Sugere-se que as células na segunda para a terceira passagem são adequados para in vitro e in vivo para estudar a biologia endotelial. As Notas-chave do Protocolo Present <p class="jove_content...

O endotélio vascular é não só uma camada de barreira que separa o sangue e os tecidos, considera-se um grande glândula endócrina que se estende ao longo de toda a árvore vascular com uma área de superfície de 400 metros quadrados 1. O bem-estar do endotélio é essencial para a homeostase vascular. O endotélio disfuncional participa em várias desordens cardiovasculares, incluindo a aterosclerose, vasculite e a lesão de isquemia / reperfusão, etc. 2-4. Até à data, os mecanismos celulares e moleculares específicos envolvidos nestas configurações de doença não são bem compreendidos, devido à natureza anatómica difusa de endotélio. O rato é um modelo importante para a pesquisa porque as técnicas de manipulação genética são mais desenvolvidos em ratos do que em quaisquer outras espécies de mamíferos. No entanto, o isolamento de células endoteliais de aorta de murino primário é considerado particularmente difícil devido ao pequeno tamanho da aorta torna enzymatIC digestão do endotélio impraticável. Alguns relataram procedimentos para isolar e purificar ECs exigem 5-7. O objetivo deste protocolo é a utilização de um método simples para isolar e expandir células endoteliais da aorta do rato sem o uso de qualquer equipamento especial. Neste protocolo, a aorta isolada de fresco é cortada em pequenos segmentos e semearam-se sobre uma matriz com o endotélio virada para baixo para permitir a germinação endotelial. Após segmentos são removidos, as células endoteliais são expandidos em forma favorecida do endotélio e está pronto para experimentos após dois ou três passagens. As vantagens do método descrito são as seguintes: 1) consideravelmente elevados números de células endoteliais são colhidas a partir de um único aorta; 2) a viabilidade das células é bem preservada; e 3) é necessário nenhum equipamento ou técnica especial. Ele fornece um meio eficaz de identificar mecanismos celulares e moleculares específicos na fisiopatologia das células endoteliais. Para aqueles que estão interessados ​​em estudar prcélulas endoteliais cultivadas imary de ambos os camundongos knock-out de genes, gene knock-in ratos, ou um modelo de doença murino, este protocolo é muito útil e fácil de praticar.

<table><tbody><tr><td>4- or 6-week-old mice</td><td>Jackson Laboratory</td><td>#000664</td><td></td></tr><tr><td>Sterile 1x phosphate-buffered saline (PBS)</td><td>Gibco</td><td>#10010-023</td><td></td></tr><tr><td>Sterile 1x PBS containing 1,000 U/mL of heparin</td><td>Sigma Aldrich</td><td>H3149</td><td></td></tr><tr><td>Endothelial cell growth medium (Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium [DMEM] with 25&amp;nbsp;mM HEPES [Gibco, #12320-032], supplemented with 100&amp;nbsp;&amp;mu;g/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000&amp;nbsp;U/mL heparin [Sigma Aldrich, H3149], 10,000&amp;nbsp;U/mL penicillin and 10&amp;nbsp;mg/mL streptomycin [Gibco, #15140-122])</td><td></td><td></td><td></td></tr><tr><td>Growth factor reduced matrix</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>Neutral proteinase (Dispase, 1&amp;nbsp;U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034&amp;nbsp;g/L, Sigma, #1255), in Dulbeccos&amp;rsquo; Modified Eagle&amp;rsquo;s Medium, low glucose (Gibco, #12320-032)</td><td></td><td></td><td></td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)</td><td>Life Technologies</td><td>L3484</td><td></td></tr><tr><td>FITC-labeled &lt;em&gt;Ulex europaeus&lt;/em&gt; agglutinin (&lt;em&gt;Ulex&lt;/em&gt;-Lectin)</td><td>Sigma</td><td>L9006</td><td></td></tr><tr><td>Anti-mouse CD31-FITC conjugated antibody</td><td>BD Biosciences</td><td>553372</td><td></td></tr><tr><td>Anti-mouse vascular endothelial growth factor receptor 2 antibody</td><td>Cell Signaling</td><td>9698</td><td></td></tr><tr><td>Anti-mouse endothelial nitric oxide synthase</td><td>Abcam</td><td>ab5589</td><td></td></tr><tr><td>Anti-mouse vascular endothelium-cadherin</td><td>Abcam</td><td>33168</td><td></td></tr><tr><td>Anti-mouse calponin</td><td>Abcam</td><td>700</td><td></td></tr><tr><td>FITC-conjugated anti-rabbit IgG</td><td>Sigma</td><td>F6005</td><td></td></tr><tr><td>1 mL syringe fitted with 25-G needle</td><td>Fisher Scientific</td><td>50-900-04222</td><td></td></tr><tr><td>100 mm Peri dishes</td><td>Fisher Scientific</td><td>07-202-516</td><td></td></tr><tr><td>Six-well cell culture plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>T12.5 culture flask</td><td>Fisher Scientific</td><td>50-202-076</td><td></td></tr><tr><td>Scissors, forceps, microdissection scissors and forceps, Scalpel blade</td><td>Fine Science Tools, Inc.</td><td></td><td></td></tr><tr><td>Anesthesia machine with isoflurane</td><td>Webster Veterianary Supply</td><td>07-806-3204</td><td></td></tr><tr><td>heating lamp</td><td></td><td></td><td></td></tr><tr><td>Centrifuge machine</td><td></td><td></td><td></td></tr><tr><td>Inverted phase-contrast microscope</td><td></td><td></td><td></td></tr><tr><td>inverted fluorescence microscope</td><td></td><td></td><td></td></tr></tbody></table>

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.

Sprouting celular endotelial células endoteliais espontânea brotando iniciado a partir de um segmento do mouse aorta. O segmento de rato aorta foi deixada crescer numa matriz reduzida pelo factor de crescimento, em seguida, em meio de crescimento de células endoteliais durante 4 dias. O surgimento de células endoteliais normalmente aparece em 2 - 4 dias. As fotomicrografias foram tomadas no dia 4 (Figura 1). Como mostrado nas figuras, numerosas células ...

Watch this Scientific Journal Video about Isolation and Primary Culture of Mouse Aortic Endothelial Cells at JoVE.com

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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-and-primary-culture-of-mouse-aortic-endothelial-cells

Research

JoVE Journal

Biology

30.5K visualizações.  Wayne State University. 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.

Vídeo: Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

Isolamento da Primária Murino Cérebro microvascular células endoteliais

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Neurociência

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 ...

Biologia

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolamento e Cultura de Expansão de células endoteliais específicos de tumores

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Medicina

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

Isolamento de células endoteliais Microvascular do principal músculo esquelético murino

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

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 ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Isolamento de Células Endoteliais Primárias de Pulmão de Camundongo

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Imunologia e Infecção

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

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 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 ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Isolamento e cultura de células endoteliais aórticas primárias de suínos em miniatura

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Isolamento e Caracterização de Células Endoteliais Hemorlicais do Fígado Primário do Rato

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...