Name: isolation and culture expansion of tumor-specific endothelial cells
Uploaded: 2015-10-14T15:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

O protocolo a seguir é realizada de acordo com as diretrizes estabelecidas pelo Departamento de Medicina Laboratorial Animal da Universidade da Carolina do Norte em Chapel Hill. 1. Prepare os seguintes materiais e reagentes Antes da partida <ol><li> Prepare meios CE, completando 400 ml de baixo teor de glucose (1 g / L de D-glucose ou LG) de Dulbecco modificado por meio Eagle (DMEM) com 50 ml de soro fetal de bovino inactivado pelo calor, 50 mL de Nu-Soro IV, 5 ml de antibiótico-antimicótico, e o hFGF, VEGF, hEGF, R3-IGF-1, heparina e os componentes do kit comercial. </li><li> Preparar 500 ml de tampão FACS (0,5% de BSA e EDTA a 2 mM em PBS); filtrar através de um estéril 0,22 um copo do filtro. </li><li> Esterilizar ou desinfectar dissecando bordo. </li><li> Esterilizar pinos dissecção, tesoura cirúrgica e dissectores. </li></ol> 2. Isolamento CE (Dia 1, ~ 5 h) <ol><li> Euthanize rato com dióxido de carbono ou outros métodos sagacidade compatívelh Comitê de Cuidados e Uso de Animais Institucional (IACUC) políticas. Nota: Múltiplos tumores mamários que variam em tamanho (5 - 15 mm de diâmetro) pode se desenvolver em um único rato geneticamente modificado, como C3-tag, certifique-se de colher todos eles para o isolamento do TCE. Se estiver usando os tumores que são ortotopicamente enxertados em ratos, piscina, duas a três um centímetro 3 tumores para um único isolamento CE. Aqui usamos tumores mamários como uma demonstração, mas o protocolo pode ser modificado para outros tipos de tumores. </li><li> Desinfetar rato por aspersão ou limpando o rato lado ventral com uma ampla quantidade de 75% v / v de etanol. </li><li> Ressecção de tumores com um par de tesouras e pinças usando técnicas assépticas em uma capa estéril ou em uma bancada limpa. <ol><li> Estique-se e fixar os membros dos ratos em uma placa de dissecação. Faça uma incisão na linha média ventral com a tesoura sem abrir o peritônio. Dissecar lateralmente entre a pele e o peritoneu para as glândulas mamárias em que os tumores são localizados.Não abra o peritônio. </li><li> Excise único tecido do tumor das glândulas mamárias, deixando de fora as margens mamárias normais. Corte com cuidado tecidos não tumorais, como a pele e músculos, e coloque tumores dissecados em um tubo cônico, contendo 30 ml de DMEM-LG no gelo. </li></ol></li><li> Trazer amostras de tumores para capa de cultura de tecidos; lavar tecidos com estéril LG-DMEM 1 - 2 vezes. </li><li> Tumores transferência do tubo cônico para um estéril cultura de tecidos de petri prato, adicione 2 ml de DMEM-LG no prato, e pique com um par de tesouras estéreis em pedaços &lt;5 mm. </li><li> Adicionar 5 ml de colagenase (banco = 2 mg / ml em solução salina equilibrada de Hank, daqui em diante HBSS), 1 ml de dispase (estoque = 2,5 U / mL em HBSS) e 75 ul de desoxirribonuclease (estoque = 1 mg / ml em PBS ) para dentro do prato de petri. O volume total é agora ~ 10 ml. </li><li> Transferir a mistura de colagenase / tecido do prato de Petri para um tubo de tecido-Dissociator e executado em um Dissociator tecido durante 60 segundos duas vezes (pré-definido de dissociação PRogram no Dissociator: 1.270 Total de Rodadas por corrida). Incubar com luz agitando num agitador durante 75 min a 37 ° C. </li><li> Filtro de tecido digerido através de um filtro de células de 100 mm ao longo de um tubo de 50 ml. Lavar o filtro com 5 ml de tampão de FACS para lavar quaisquer células restantes. Girar a 280 xg durante 5 min e o sobrenadante foi cuidadosamente aspirado sem perturbar o sedimento celular. </li><li> Dilui-se 1 ml de tampão de lise de RBC estoque (10x) em 9 ml de água estéril. Lisar células vermelhas do sangue com 10 ml de tampão de lise (1x), e imediatamente girar 5 min a 280 x g. Nota: Esta etapa pode ser ignorada se pouco sangue é visível. </li><li> Ressuspender em 10 ml de tampão de FACS. Misturar 10 ul de suspensão celular com 10 ul de azul de tripano e contagem de células vivas utilizando um hemocitómetro. </li><li> Ressuspender as células em 10 ~ 7 células / 100 uL. Adicionar 10 ul de FcR bloco por 100 ul de suspensão de células, e incuba-se em gelo durante 10 min. </li><li> Adicionar anticorpo CD31 anti-rato rato conjugado com PE de acordo. a Tabela 1 Incubar em gelo durante 10 - 15 minutos e o tubo de filme ocasionalmente. </li><li> Adicionar 10 ml de tampão de SCAF e para o tubo de centrifugação a 280 xg durante 5 min. Remover o sobrenadante cuidadosamente, e lava-se a pelete de células novamente com 5 mL de tampão de FACS. Centrifugar para sedimentar as células. Aspirar o sobrenadante sem perturbar sedimento celular. </li><li> Adicionar tampão e anti-PE FACS microesferas de acordo com a Tabela 2 Incubar em gelo durante 10 -. 15 min. Tubo Flick ocasionalmente. </li><li> Adicionar 10 ml de tampão de amostras e de rotação a 280 xg FACS durante 5 min; lavar uma vez com 5 mL de tampão de FACS e centrifugar novamente. Aspirar o sobrenadante sem perturbar pellet. </li><li> Levar o volume até 300 ml em tampão de SCAF. Gire a 35 mm de células-filtro tampado tubo 5 min a 280 x g. Utilize tubos 2 e um volume maiores, se necessário. </li><li> Configurar multistand magnética e colunas magnética na capa, uma coluna para fixar o separador e equilibrar a coluna com 2 mL de tampão de FACS. </li><li> Aspirate o sobrenadante e ressuspender o sedimento de células em 0,5 - 1 ml de tampão de FACS. </li><li> Passar a suspensão de células através de uma coluna magnética equilibrada. </li><li> Lava-se a coluna três vezes com 2 ml de tampão de FACS, e recolher o fluxo de passagem (FT) em um tubo de 15 ml (fracção FT). </li><li> Aqui a coluna fora do separador e elui-se com 2 mL de tampão de FACS para outro tubo de 15 ml (fracção de eluato). Use êmbolo para assegurar que todas as células estão a sair da coluna. Repetir a eluição duas vezes mais com 2 ml de tampão de FACS de cada vez. </li><li> Girar o eluato a 280 xg durante 5 min. </li><li> Remover o sobrenadante e ressuspender o sedimento em 10 ml de meio de CE. </li><li> Igualmente dividir a fracção de eluato (~ 6 ml) em placas revestidas com gelatina de 10 cm. Para três tumores 1 3 cm, placa de células eluída em pelo menos quatro placas. Células Alternativamente, placa eluidas a diferentes concentrações em várias placas (por exemplo., De sementes de 0,5 ml, 1 ml, 1,5 ml e 3 ml do eluato em quatro placas) para garantir que umt menos uma placa de baixa densidade é semeada com células eluídas. Verificar que a confluência das células ligadas é a ~ 1% no dia seguinte (ou seja, aproximadamente 1,0 - 2,0 x 10 5 células ligadas). Nota: As células têm de ser banhado escassa, de forma que podem formar colónias CE sem ser contaminado por outros tipos de células. </li><li> Placa fração FT em uma placa de 10 cm para permitir que as células recuperar O / N. Verificar que a placa é de 80 - 100% confluentes no dia seguinte. Congelar-se as células (-80 ° C) em 250 meios de congelamento de células em um ul cryotube e armazená-los em nitrogênio líquido no dia seguinte. Nota: fracção FT pode ser usado para isolamento de células de tumor numa fase posterior, e / ou como um controlo negativo para a análise dos clones isolados a expressão do gene da CE CE. </li><li> Mudança de mídia a cada 2 - 3 dias. Colónias começam a se formar após 7 - 10 dias. As pequenas colónias CE pode ser identificado como o mais cedo dia 3. Marcar as colónias com um marcador de ponta fina na parte inferior do prato. </li><li> Raspar não específica cells que cercam as colônias identificadas com um estéril 200 ul pippet ponta. </li></ol> 3. Colony Seleção Anéis Usando Clonagem <ol><li> Mudança de mídia a cada 2 - 3 dias. CE pureza pode ser verificada por LDL (DII-Ac-LDL) além de cerca de 5-7 dias. Adicionar 50 ul de LDL por 10 ml de meio CE e incubar por 3-4 horas antes de verificar as células em microscópio de fluorescência. Nota: LDL Múltipla + clones CE pôde ser observada a ~ 7 dias. </li><li> Comece a colheita colônias CE quando atingem diâmetros de 3 - 5 mm de tamanho. (Escolha grandes colônias que são embalados com pequenas células de LDL + para obter melhores resultados.) </li><li> Antes da colheita CE com anéis de clonagem, pré-coat algumas placas de 6 poços com 0,5% de gelatina. Aspirar gelatina, adicionar 2 ml de meio CE a cada poço, e manter as placas em uma incubadora até que seja necessário. </li><li> Raspar não-CE nas bordas das colónias para se certificar não há outros tipos de células irá ficar preso entre o anel de clonagem. </li><li> Usando ummicroscópio de contraste de fase (4x ou 10x objectivo), o contorno com um marcador de ponta fina na parte inferior da placa de cultura as áreas que continham colónias CE. </li><li> Lava-se a placa com 10 ml de PBS e deixar uma camada muito fina de PBS na placa quando a aspiração. (Importante: uma pequena quantidade (~ 0,5 ml) de PBS vai manter as células vivas durante o procedimento de clonagem de anel, também, adesivo de tecido precisa de água para se relacionar.) </li><li> Escolha um anel de clonagem de tamanho apropriado. Use um par de pinças de dissecação para pegar um anel, e com uma pipeta de ponta 10 ul aplicar uniformemente uma pequena quantidade de adesivo tecidual sobre o anel de clonagem. Nota: usar apenas uma quantidade mínima (~ 0,2 mL para um pequeno anel) de adesivo de tecido em anéis de clonagem, e fazer adesivo de tecido certeza é se espalhar uniformemente ao redor da superfície do fundo para garantir uma boa vedação. Adesivo tecidual excessiva produz calor e faz filmes que podem matar as células. </li><li> Coloque o anel de clonagem sobre a colônia CE. Pressione suavemente para baixo o anel de clonagem para colar as ring sobre a placa. Certifique-se de que as colônias não estão secas antes de colar o anel. </li><li> Imediatamente pipeta 25 ul de solução de separação celular enzimática para dentro do anel de clonagem e incubar ~ 1 min ou até que as células são frouxamente ligado. </li><li> Pipetar as células no anel clonagem gota a gota, para um poço de uma placa de 6 poços contendo meio CE pré-aquecido. (Importante: Não agite a placa para dispersar as células; CE preferem crescer em clusters apertados.) Lave o anel de clonagem com 50 - 100 media CE ul para coletar tantas células quanto possível, e transferir todas as lavagens na mesma 6 poços . </li><li> Se algumas colônias na placa de 10 cm são demasiado pequenas para colher, adicionar 10 ml de mídia fresco, deixar as colônias crescer por mais alguns dias, e repita o procedimento de clonagem anel novamente. </li><li> Cresça colhidas colónias em placas de 6 poços até 80 - 100% confluentes, e transferência de células a 3 poços de uma placa de 6 cavidades, antes de se expandir-los em uma 10 cm prato de cultura de tecidos. Raspe as células contaminantes. Repitaoutra rodada de procedimento anel de clonagem (Passos 3,5-3,10), se necessário. Mantenha as células relativamente confluente (~ 60 - 70%) quando se expandindo. CE pode parar de crescer, se banhado muito escassa. </li><li> Caracterizar CE por FACS, coloração, PCR, etc. Note que Dil-Ac-LDL é fluorescente e pode interferir com PE ou outros anticorpos fluorescentes para FACS. </li></ol>

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Path. 194 (2), 254-261 (2001).</li><li>Maddaluno, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Cooley, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling+mediates+endothelial-to-mesenchymal+transition+(EndMT)+during+vein+graft+remodeling.">TGF-&#946; signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.</a> Sci. Transl. Med. 6 (227), 227ra34-227ra34 (2014).</li><li>Garcia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie1+deficiency+induces+endothelial-mesenchymal+transition.">Tie1 deficiency induces endothelial-mesenchymal transition.</a> EMBO Rep. 13 (5), 431-439 (2012).</li><li>Xiao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+endothelial+cells+with+distinct+patterns+of+TGF&#946;-driven+endothelial-to-mesenchymal+transition.">Tumor endothelial cells with distinct patterns of TGF&#946;-driven endothelial-to-mesenchymal transition.</a> Cancer Res. 75 (7), 1244-1254 (2015).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+clonally+expanding+haematopoietic+compartment+in+bone+marrow.">Identification of a clonally expanding haematopoietic compartment in bone marrow.</a> EMBO J. 32 (2), 219-230 (2012).</li><li>Sawamiphak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin-B2+regulates+VEGFR2+function+in+developmental+and+tumour+angiogenesis.">Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.</a> Nature. 465 (7297), 487-491 (2010).</li><li>Chi, J. -. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+diversity+revealed+by+global+expression+profiling.">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</li><li>Nolan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev. Cell. 26 (2), 204-219 (2013).</li><li>Ingram, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li></ol>

The authors have nothing to disclose.

Devido às dificuldades na obtenção de culturas puras TEC primária, muitos estudos in vitro TEC substituto com linhas CE comercialmente disponíveis ou CE primário, como veia umbilical humano CE (HUVEC) 13. No entanto, essas populações da CE a partir de tecidos normais só podem servir como uma proxy para a TEC que diferem marcadamente dos seus homólogos normais. Por exemplo, a TEC são fenotipicamente e funcionalmente anormal in vivo e algumas destas alterações podem ser transmiss?...

Células endoteliais (EC) são essenciais durante o desenvolvimento de tumores sólidos. Do início do interruptor angiogênico em tumores dormentes para divulgação e propagação de metástases em locais distantes, CE constituem as condutas que fornecem sangue, oxigênio e nutrientes para sustentar o crescimento do tumor 1. Como sugerido recentemente, CE também têm funções independentes de perfusão e formar um nicho que suporta o crescimento de células-tronco cancerígenas e outras células do estroma do tumor 2-5. Assim, altamente purificado CE (TEC) para a cultura in vitro específicos do tumor permite estudos funcionais de rotina que irá lançar luz sobre os mecanismos moleculares que medeiam a angiogênese do tumor e conversas cruzadas com células tumorais. CE são altamente especializados, dependendo do tecido de origem 6. Devido à natureza heterogénea de diferentes tipos de tumores e o microambiente do tumor, TEC também pode exibir características únicas que reflectem uma especialização o específico de tumorf a vasculatura. Por exemplo, existe uma variabilidade notável nas assinaturas de expressão de genes em TEC isoladas de diferentes tipos ou graus de tumores 7,8. No entanto, freqüente co-purificação de não-CE, especialmente fibroblastos associados a tumores e células tumorais, com TEC pode confundir expressão de todo o genoma analisa. Estes tipos de células não desejadas são especialmente problemáticos em estudos que dependem de longo prazo expansão in vitro de culturas TEC. Aqui descrito é um método de alta fidelidade, que produz consistentemente culturas puras CE de tumores e outros tecidos. Seguindo imunomagnï¿½ica enriquecimento de frações da CE e remoção de co-purificado não-CE coluna, um passo de clonagem-ring adicional para capturar colônias CE puros é usado 9. Cada colônia podem ser expandidas em cultura para várias passagens sem o surgimento de contaminação não-CE. Este método também produz vários clones de CE de um único procedimento de isolamento, o qual é ideal para o estudo de endoheterogeneidade endotelial. Além disso, é mostrado que Cdh5 cre: / l ratos ZsGreen l / s repórter são uma ferramenta valiosa para gerar CE &quot;mapeado-destino&quot; e indelevelmente marcadas que manter ZsGreen fluorescência na cultura 10. Com pequenos ajustes para o protocolo, este método deve ser adaptável a diferentes tipos de tumores ou tecidos normais.

<table><tbody><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s medium (1 g/L D-glucose) (LG-DMEM)</td><td>Gibco</td><td>11885-084</td><td></td></tr><tr><td>EGM-2 Bullet Kit&amp;nbsp;</td><td>Lonza</td><td>CC4176</td><td>Not all components used</td></tr><tr><td>Fetal bovine serum (Hyclone)</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Heat inactivated at 56 &amp;deg;C for 30 min</td></tr><tr><td>Nu-Serum IV</td><td>Corning</td><td>CB-51004</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBBS)</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td></td><td></td><td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &amp;mu;m filter</td></tr><tr><td>75% v/v ethanol for disinfection</td><td></td><td></td><td></td></tr><tr><td>Anti-PE microbeads&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-048-801</td><td></td></tr><tr><td>Bovine serum albumin (BSA) fraction V, 7.5%</td><td>Gibco</td><td>15260-37</td><td></td></tr><tr><td>Cell freezing media (Bambanker)</td><td>Wako Chemicals</td><td>302-14681</td><td></td></tr><tr><td>Collagenase type II&amp;nbsp;&amp;nbsp;</td><td>Worthington Biochemical</td><td>LS004176</td><td>Make stock concentration 2 mg/ml in HBSS</td></tr><tr><td>Deoxyribonuclease I (DNase)</td><td>Worthington Biochemical</td><td>LS002004</td><td>Make stock concentration 1 mg/ml in PBS</td></tr><tr><td>Dil-Ac-LDL</td><td>Biomedical Technologies</td><td>BT-902</td><td></td></tr><tr><td>EDTA, 0.5M, pH 8.0</td><td>Cellgro</td><td>46-034-CL</td><td></td></tr><tr><td>Enzymatic cell detachment solution (Accutase)</td><td>Sigma-Aldrich</td><td>A6964-100ML</td><td></td></tr><tr><td>Gelatin, 2% in water, tissue culture grade</td><td>Sigma-Aldrich</td><td>G1393-100ML</td><td>Dilute in PBS to make 0.5% gelatin solution</td></tr><tr><td>Mouse FcR Blocking Reagent&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-092-575</td><td></td></tr><tr><td>Neutral protease (Dispase)</td><td>Worthington Biochemical</td><td>LS02104</td><td>Make stock concentration 2.5 U/ml in HBSS</td></tr><tr><td>PE-rat anti-mouse CD31 antibody</td><td>BD Pharmingen</td><td>553373</td><td></td></tr><tr><td>RBC lysis buffer (BD Pharm Lyse)</td><td>BD Pharmingen</td><td>555899</td><td></td></tr><tr><td>Sterile water</td><td></td><td></td><td></td></tr><tr><td>Trypan blue, 0.4 %&amp;nbsp;</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>10 mm tissue culture dishes</td><td>Corning</td><td></td><td></td></tr><tr><td>15 ml conical tubes (sterile)</td><td>Corning</td><td></td><td></td></tr><tr><td>50 ml conical tubes (sterile)&amp;nbsp;</td><td>Corning</td><td></td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Corning</td><td></td><td></td></tr><tr><td>Tissue-dissociator tubes (gentleMACS) C tubes)&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-237</td><td></td></tr><tr><td>Cell Separator&amp;nbsp; (MidiMACS)</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>Cell strainer 100 &amp;mu;m&amp;nbsp;</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>Cloning rings (assorted sizes)</td><td>Bel-Art Products</td><td>378470000</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Dissecting board</td><td></td><td></td><td>Sterilize or disinfect with 75% v/v ethanol before use&amp;nbsp;</td></tr><tr><td>Dissecting forceps and scissors</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>Dissecting pins 2&quot;</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>FACS tubes with 35 &amp;mu;m filter cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Filter cup (Stericup, 0.22 &amp;mu;m)</td><td>Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>Fine-tip marker</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>Magnetic Multistand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>Tissue adhesive (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>Or a centrifuge with similar capacity for 15 ml and 50 ml conical tube centrifugation</td></tr><tr><td>Tissue culture hood</td><td></td><td></td><td></td></tr><tr><td>Tissue dissociator (gentleMACS)</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Preset program &quot;m_impTumor_01&quot; used for tissue dissociation&amp;nbsp;</td></tr><tr><td>Liquid nitrogen freezer</td><td></td><td></td><td></td></tr><tr><td>Microplate or rotary shaker</td><td></td><td></td><td></td></tr><tr><td>Phase contrast light microscope</td><td></td><td></td><td></td></tr></tbody></table>

isolation and culture expansion of tumor-specific endothelial cells

Células específicas do tumor isoladas de fresco endoteliais (TEC) pode ser usada para explorar mecanismos moleculares de angiogénese tumoral e servem como um modelo in vitro para o desenvolvimento de novos inibidores da angiogénese para o cancro. No entanto, a longo prazo expansão in vitro de células endoteliais de murino (CE) é um desafio devido à deriva fenotípica em cultura (transição endotelial-a-mesenquimal) e contaminação com não-CE. Isto é especialmente verdadeiro para o TEC que são prontamente outcompeted por fibroblastos co-purificada ou células de tumor em cultura. Aqui, um método de isolamento de alta fidelidade, que tira vantagem de enriquecimento imunomagnética acoplada com selecção de colónias e expansão in vitro é descrita. Esta abordagem gera frações CE puros que são totalmente livre de contaminação de células do estroma ou tumorais. Também é mostrado que traçou-linhagem Cdh5 CRE: ratinhos repórter / l ZsGreen L / S, utilizada com o protocolo aqui descrito, são uma ferramenta valiosa para verificar a célulapureza como as colônias isoladas CE destes ratos mostram durável e brilhante fluorescência ZsGreen em cultura.

CE representam apenas uma fracção pequena da população total de células na maioria dos tecidos adultos 11. É, portanto, importante para digerir completamente o tecido colhido numa suspensão de célula única que assegura a libertação máxima de CE de matriz extracelular (ECM) e tecidos conjuntivos. Em nossa experiência, a seleção imunomagnï¿½ica mediada por CD31 só fornece frações CE enriquecidos mas não puros; portanto, um passo crucial é a remoção física de co-purificado não-CE e sel...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

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 ...

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

Research

JoVE Journal

Medicine

11.1K visualizações.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

Vídeo: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Expansão ex vivo de células tumorais T-reativa por Meios de 1/Ionomycin Bryostatin eo Common Gamma Cadeia Formulação Citocinas

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Imunologia e Infecção

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Isolamento de células endoteliais humanas do cólon Normal e Carcinoma colorrectal - um protocolo melhorado

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Pesquisa do câncer

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Isolamento de células progenitoras endoteliais de sangue humano do Cordão Umbilical

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Biologia do Desenvolvimento

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Isolamento de células do cólon tumor primário Humanos a partir de tecidos cirúrgicos e cultivando-as Diretamente macias elásticas Substratos para Traction Citometria

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Bioengenharia

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

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

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

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

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

Biologia

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Neurociência

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

Um modelo de xenoenxerto derivado do paciente para malformação venosa

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

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 of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Isolamento de CD133 + células tronco do fígado de Expansão Clonal

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.