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>

<ol>
	<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-angiogenesis:+new+concept+for+therapy+of+solid+tumors.">Anti-angiogenesis: new concept for therapy of solid tumors.</a> Ann. Surg. 175 (3), 409-416 (1972).</li><li>Butler, J. M., Kobayashi, H., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instructive+role+of+the+vascular+niche+in+promoting+tumour+growth+and+tissue+repair+by+angiocrine+factors.">Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors.</a> Nat. Rev. Cancer. 10 (2), 138-146 (2010).</li><li>Franses, J. W., Baker, A. B., Chitalia, V. C., Edelman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+endothelial+cells+directly+influence+cancer+progression.">Stromal endothelial cells directly influence cancer progression.</a> Sci. Transl. Med. 3 (66), 66ra5 (2011).</li><li>Calabrese, 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=A+perivascular+niche+for+brain+tumor+stem+cells.">A perivascular niche for brain tumor stem cells.</a> Cancer Cell. 11 (1), 69-82 (2007).</li><li>Beck, B., 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=A+vascular+niche+and+a+VEGF-Nrp1+loop+regulate+the+initiation+and+stemness+of+skin+tumours.">A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours.</a> Nature. 478 (7369), 399-403 (2011).</li><li>Aird, W. C. <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+heterogeneity.">Endothelial cell heterogeneity.</a> Cold Spring Harb. Perspect. Med. 2 (1), a006429 (2012).</li><li>Dudley, A. C. <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.">Tumor endothelial cells.</a> Cold Spring Harb. Perspect. Med. 2 (3), a006536-a006536 (2012).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+heterogeneity+of+tumor+endothelium.">Molecular heterogeneity of tumor endothelium.</a> Cell Tissue Res. 335 (1), 271-281 (2009).</li><li>Voyta, J. C., Via, D. P., Butterfield, C. E., Zetter, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+isolation+of+endothelial+cells+based+on+their+increased+uptake+of+acetylated-low+density+lipoprotein.">Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein.</a> J. Cell Biol. 99 (6), 2034-2040 (1984).</li><li>Zovein, A. 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=Fate+tracing+reveals+the+endothelial+origin+of+hematopoietic+stem+cells.">Fate tracing reveals the endothelial origin of hematopoietic stem cells.</a> Cell Stem Cell. 3 (6), 625-636 (2008).</li><li>Beijnum, J. R., Rousch, M., Castermans, K., van der Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. Protoc. 3 (6), 1085-1091 (2008).</li><li>Xiao, L., Harrell, J. C., Perou, C. M., Dudley, A. C. <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+stable+molecular+signature+in+mammary+tumor+endothelial+cells+that+persists+in+vitro.">Identification of a stable molecular signature in mammary tumor endothelial cells that persists in vitro.</a> Angiogenesis. 17 (3), 511-518 (2014).</li><li>Beijnum, J. R., 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=Gene+expression+of+tumor+angiogenesis+dissected:+specific+targeting+of+colon+cancer+angiogenic+vasculature.">Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature.</a> Blood. 108 (7), 2339-2348 (2006).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nat. Med. 9 (6), 713-725 (2003).</li><li>Baluk, P., Hashizume, H., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+abnormalities+of+blood+vessels+as+targets+in+cancer.">Cellular abnormalities of blood vessels as targets in cancer.</a> Curr. Opin. Genetics Dev. 15 (1), 102-111 (2005).</li><li>Ghosh, K., 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-derived+endothelial+cells+exhibit+aberrant+Rho-mediated+mechanosensing+and+abnormal+angiogenesis+in+vitro.">Tumor-derived endothelial cells exhibit aberrant Rho-mediated mechanosensing and abnormal angiogenesis in vitro.</a> Proc. Natl. Acad. Sci. 105 (32), 11305-11310 (2008).</li><li>Amin, D. N., Hida, K., Bielenberg, D. R., Klagsbrun, M. <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+express+epidermal+growth+factor+receptor+(EGFR)+but+not+ErbB3+and+are+responsive+to+EGF+and+to+EGFR+kinase+inhibitors.">Tumor endothelial cells express epidermal growth factor receptor (EGFR) but not ErbB3 and are responsive to EGF and to EGFR kinase inhibitors.</a> Cancer Res. 66 (4), 2173-2180 (2006).</li><li>Hida, K., 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-associated+endothelial+cells+with+cytogenetic+abnormalities.">Tumor-associated endothelial cells with cytogenetic abnormalities.</a> Cancer Res. 64 (22), 8249-8255 (2004).</li><li>Dudley, A. 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=Calcification+of+multipotent+prostate+tumor+endothelium.">Calcification of multipotent prostate tumor endothelium.</a> Cancer Cell. 14 (3), 201-211 (2008).</li><li>Dunleavey, J. M., 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=Vascular+channels+formed+by+subpopulations+of+PECAM1(+)+melanoma+cells.">Vascular channels formed by subpopulations of PECAM1(+) melanoma cells.</a> Nat. Comm. 5, 5200 (2014).</li><li>St Croix, B., 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=Genes+expressed+in+human+tumor+endothelium.">Genes expressed in human tumor endothelium.</a> Science. 289 (5482), 1197-1202 (2000).</li><li>Bhati, R., 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+characterization+of+human+breast+tumor+vascular+cells.">Molecular characterization of human breast tumor vascular cells.</a> Am. J. Pathol. 172 (5), 1381-1390 (2008).</li><li>Johnson, C. S., Chung, I., Trump, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+CYP24+in+the+tumor+microenvironment.">Epigenetic silencing of CYP24 in the tumor microenvironment.</a> J. Steroid Biochem. Mol. Biol. 121 (1-2), 338-342 (2010).</li><li>Gimbrone, M. A., Cotran, R. S., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+vascular+endothelial+cells+in+culture.+Growth+and+DNA+synthesis.">Human vascular endothelial cells in culture. Growth and DNA synthesis.</a> J. Cell Biol. 60 (3), 673-684 (1974).</li><li>Burridge, K. A., Friedman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+and+vascular+bed+origin+influence+differences+in+endothelial+transcriptional+profiles+of+coronary+and+iliac+arteries.">Environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299 (3), H837-H846 (2010).</li><li>Zhang, J., Burridge, K. A., Friedman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+differences+between+endothelial+transcriptional+profiles+of+coronary+and+iliac+arteries+revealed+by+microarray+analysis.">In vivo differences between endothelial transcriptional profiles of coronary and iliac arteries revealed by microarray analysis.</a> Am. J. Physiol. Heart Circ. Physiol. 295 (4), H1556-H1561 (2008).</li><li>Paruchuri, 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=Human+pulmonary+valve+progenitor+cells+exhibit+endothelial/mesenchymal+plasticity+in+response+to+vascular+endothelial+growth+factor-A+and+transforming+growth+factor-beta2.">Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2.</a> Circ. Res. 99 (8), 861-869 (2006).</li><li>Wylie-Sears, J., Aikawa, E., Levine, R. A., Yang, J. -. H., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitral+valve+endothelial+cells+with+osteogenic+differentiation+potential.">Mitral valve endothelial cells with osteogenic differentiation potential.</a> Arterioscler. Thromb. Vasc. Biol. 31 (3), 598-607 (2011).</li><li>Ginsberg, M., 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=Efficient+direct+reprogramming+of+mature+amniotic+cells+into+endothelial+cells+by+ETS+factors+and+TGF&#946;+suppression.">Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGF&#946; suppression.</a> Cell. 151 (3), 559-575 (2012).</li><li>Sapino, 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=Expression+of+CD31+by+cells+of+extensive+ductal+in+situ+and+invasive+carcinomas+of+the+breast.">Expression of CD31 by cells of extensive ductal in situ and invasive carcinomas of the breast.</a> J. 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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;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&#160;</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&#176;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&#39;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&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &#956;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&#160;</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&#160;&#160;</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&#160;</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 %&#160;</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)&#160;</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)&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator&#160; (MidiMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 &#956;m&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Dissecting forceps and scissors</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting pins 2&#34;</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>FACS tubes with 35 &#956;m filter cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cup (Stericup, 0.22 &#956;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 &#34;m_impTumor_01&#34; used for tissue dissociation&#160;</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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolamento, cultura, e imagem de Fetal Humano pancreáticas grupos de células

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Isolamento, criopreservação e cultivo de células epiteliais humanas Amnion para aplicações clínicas

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolamento de linfático humano endotelial por multi-parâmetro activadas por fluorescência celular

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolamento e cultura de células endoteliais primárias de Artérias e Veias canina

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Isolamento de células endoteliais progenitoras de voluntários saudáveis ​​e seu potencial migratório influenciada por amostras de soro após cirurgia cardíaca

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

preparação e<em&gt; In Vitro</em&gt; Caracterização de magnetizado miR-modificado Células Endoteliais

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

Avaliação das propriedades das células endoteliais utilizando cultura de células 3D e sangue não anticoagulado anticoagulantes e anti-inflamatório

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Desequilíbrios musculares: Teste e treinamento de força funcional da limitação excêntrico em populações atléticas

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...