Os autores agradecem Katharina Schallmoser para fornecer pHPL, Eva Rohde e Nicole Hofmann por seus úteis comentários ao manuscrito, Daniela Thaler e Margaretha Fr hwirth para assistência técnica, Monica Farrell para edição linguística, Uwe Lang, Margit Holzapfel e Sandra Eppich no Departamento de Obstetrícia para fornecer os cabos. Este trabalho tem sido apoiada pelo austríaco Research Foundation (FWF, conceder N211-NAN para DS), a Agência de Promoção austríaco Research (FFG, conceder N200 para DS) e pelo adulto Stem Cell Research Foundation (AR). AR é um companheiro da Medicina Molecular programa de doutorado da Universidade Médica de Graz.

Parte 1: Configurando 1. Preparação de meio de cultura celular Antes de iniciar a preparação de médio recolher todos os materiais e ferramentas que você precisa. Descongelar 2 x 56 ml alíquotas de pool de lisado de plaquetas humanas (pHPL, self-made: referência 1 &amp; JOVE # 1523), 10 ml de uma penicilina 100x / solução estreptomicina, 2 x 5 mL de L-Glutamina (ambos Sigma). Descongele a citocina e, as alíquotas do fator de crescimento (VEGF, bFGF, EGF, IGF, hidrocortisona, ácido ascórbico, Heparina, Anfotericina fornecido como &#39;quots single&#39;) ajustados para completar 1 garrafa (500 ml) de EGM-2 médios (Lonza). Da geladeira tomar uma garrafa de 500 ml cada um dos (i) alfa-modificado meio essencial mínimo (a-MEM) e (ii) médio endotelial basal (EBM). Todos os passos seguintes são executadas em um fluxo laminar de cultura de tecidos em condições estéreis. Prepare heparina sem conservantes (por exemplo, Biochrom) pela dissolução do pó para uma concentração final de 1000 UI / ml com água estéril. MSC Médio: Use 500 mL de uma MEM, adicione 56 ml de pHPL descongelados (ver também referência 1 para mais detalhes) e 2 UI / mL (= 224 ml de solução-estoque) de heparina sem conservantes (evita a coagulação do meio através do acúmulo de o fibrinogênio no plasma) para alcançar uma concentração final de pHPL 10%. Além disso adicionar penicilina (100U/ml) / estreptomicina (100μg/mL) e solução de 2mM de L-glutamina (ambos Sigma). Filtrar o meio através do filtro a vácuo a 20 mM de poros tamanho (Millipore). Rótulo do frasco adequadamente (data, conteúdo). ECFC-Medium: Use uma garrafa (500 ml) de EBM, adicionar as alíquotas de citocinas, 56 mL de pHPL, 10 UI / ml (= 1120μl da solução de reserva) de conservante livre de Heparina, Penicilina (100U/ml) / estreptomicina (100 g / mL de solução) e 2mM de L-glutamina ao meio basal e com um filtro de 20 l de poros tamanho vácuo filtro (Millipore). Rótulo do frasco adequadamente (data, conteúdo). 2. Esterilização de instrumentos cirúrgicos Fórceps, tesoura afiada cirúrgica e deve ser titular bisturi calor descartáveis ​​esterilizados minério estéril. 3. Preparação de tubos para coleta de medula Adicionar 500 UI de conservante - heparina livre para 50 tubos de polipropileno ml (Falcon) e ajustar a um volume final de 20 mL com PBS. Transferir os tubos para a sala de parto. 4. Consentimento informado (confirmado por seu comitê de ética local ou IRB). Pergunte aos pais antes da entrega, se quiser doar um pedaço do cabo e informá-los adequadamente dos propósitos que irá servir. 5. Doação de medula Após a entrega e fiscalização da placenta e do cordão cortou um pedaço de 10 cm e imediatamente transferir para o tubo pré-carregada com heparina para evitar coagulação do sangue remanescente no interior dos vasos do cordão umbilical. Continuar com o isolamento de células mais rapidamente possível. Parte 2: Célula de isolamento <ol><li> Prepare câmara de fluxo laminar de cultura de tecido de limpeza de superfícies com Incidin ou 70% EtOH. Lugar estéril 150 centímetros 2 placas de cultura de células, 75 cm frascos de cultura de células (ambos Corning), PBS, instrumentos cirúrgicos esterilizados, raspadores celular, suporte para tubos, 25 stripettes mL e 5 seringas ml equipados com agulhas final blunt adequadamente em sua capa limpa. </li><li> Pré-aquecer o seu preparada α-MEM e EGM-2 meio de cultura celular para 37 ° C em banho-maria. </li><li> Trazer o cabo da sala de parto para o seu laboratório equipado com o capô de cultura de células. Depois de transferir o cabo no fluxo laminar lavá-la duas vezes em PBS (por transferência para um novo prato) para se livrar de contaminar células do sangue. Lavar a veia umbilical após canulating-lo de um lado com uma seringa de 5 mL estéril equipada com uma agulha sem corte final com PBS até que o líquido que sai do outro lado do cabo torna-se completamente transparente (avermelhada indica a contaminação do sangue). </li></ol> ECFCs: <ol start="4"><li> Prepare a 75 cm 2 frasco (Corning), rotulá-la e preencher 15-20 mL de pré-aquecido médio EGM-2. </li><li> Coloque o cabo em um novo prato cheio de PBS estéril. Pegue a tesoura cirúrgica e cortar o cordão umbilical em dois pedaços de cerca de 5 cm de comprimento (peças curtas são mais fáceis de processo, porque os vasos umbilicais são torcidas ao longo de seu eixo). </li><li> Tentar encontrar a veia umbilical (vaso grande), coloque uma lâmina de sua tesoura cirúrgica e cortar a veia longitudinalmente. Neste ponto ele poderia ser útil se um assistente pode manter a veia já cortados com duas pinças quando você cortar ainda mais. </li><li> Coloque a torta de medulace com a veia cortada em um novo prato, sem PBS, tomar o raspador de células e raspe a superfície (endoteliais) interior da veia umbilical por decalques de um lado para o outro. Transferência das células para o seu frasco de preparado por limpar a ponta do raspador no meio. Repita este procedimento pelo menos 10 vezes. </li><li> Fechar o frasco e colocá-lo em uma incubadora (37 ° C, 5% CO 2, 95% de umidade). </li></ol> CTMs: <ol start="4"><li> Prepare uma placa de cultura 150 centímetros 2 (Corning) e rotulá-la. </li><li> Pegue o pedaço de corda (depois de raspar a camada endotelial) e cortar o cordão inteiro em pedaços muito pequenos, usando uma tesoura esterilizada e bisturi estéril. Tamanho máximo das peças cabo deve ser de 1-2 mm. </li><li> Transferência de peças do seu cordão umbilical com a pinça estéril em sua placa de cultura pré-rotulados. Ao deixar a placa aberta por 5 minutos antes de encher com o meio, o anexo das peças para a superfície de plástico é fortalecida. </li><li> Uma vez que as peças cabo está conectado adequadamente ao plástico, gentilmente adicionar em 30-35 mL de pré-aquecido alfa modificado MEM. Tente usar a velocidade mais baixa para ter certeza de que todas as peças permanecem em anexo. </li><li> Feche a placa e colocá-lo em uma incubadora (37 ° C, 5% CO 2, 95% de umidade). </li></ol> Parte 3: expansão da célula e confirmação do fenótipo-imune por citometria de fluxo Expansão de células EFCFs: <ol><li> Verificar a fixação das células endoteliais no dia seguinte em um microscópio invertido e troca de toda EGM-2 médio para se livrar de todas as células não-inscritos e partículas. </li><li> Expandir as células, até confluência e de intercâmbio de um terço da média duas vezes por semana. Quando confluência é atingido, retire as células com 0,05% Trypsin/0.7 mM EDTA e transferi-los aos navios nova cultura. Dependendo do número de células dentro do seu frasco T75, você deve escolher o tamanho eo número de seu novo frascos para garantir a densidade de semeadura baixo para uma maior expansão. </li><li> Troca de um terço da média duas vezes por semana durante a expansão das células. </li></ol> CTMs: <ol><li> A conseqüência de CTMs da medula vai levar mais tempo, então você pode esperar pelo menos 3-4 dias até que você verificar em um microscópio invertido para o aparecimento de células fusiformes nos arredores das peças de tecido em anexo. Quando há células suficientes para o crescimento, as peças tendem a destacar porque eles perdem contato com o plástico. </li><li> Depois de 10-12 dias retire os pedaços de tecido, retire as CTMs do plástico com 0,05% Trypsin/0.7 mM EDTA e transferi-los aos navios nova cultura. Dependendo da quantidade de células dentro de seu prato, você deve decidir o tamanho eo número de seu novo frascos para garantir a densidade de semeadura baixo para uma maior expansão. </li><li> Troca de um terço da média duas vezes por semana durante a expansão das células. </li></ol> Citometria de fluxo Equipamento necessário: Citômetro de fluxo (BD FACSCalibur), micronic tubos (Corning), suporte do tubo, ovelhas soro (bloqueio de reagentes), Eppifuge (Eppendorf), os anticorpos monoclonais MSCs: CD45, CD14, CD19, HLA-DR, CD31, CD73, CD90, CD105, controles isotipo ECFCs: CD45, CD14, CD19, HLA-DR, CD31, CD34, CD90, CD105, CD144, CD146, controles isotipo <ol><li> Depois de atingir confluência, retire a célula com 0,05% Trypsin/0.7 mM EDTA e centrifugar a 300 g por 7 min a 4 ° C. </li><li> Ressuspender as células a uma concentração de 1x10 6 células / mL em PBS modificado e ligação do anticorpo inespecífico bloco com 10% v / v ovelhas soro por 20 min a 4 ° C. </li><li> Identifique os tubos micronic e adicione a concentração adequada de anticorpos fluorescentes marcados. </li><li> Adicionar 50 ul da suspensão celular (bloqueado) a cada tubo e incubar por 30 min a 4 ° C. </li><li> Lave as células modificadas pela adição de PBS para remover os anticorpos não vinculativa. </li><li> Ir com o seu celular rotulados para o citômetro de fluxo e realizar a análise. </li></ol> Parte 4: ECFC colônia de crescimento, a fixação coloração e análise de hierarquia <ol><li> A semente ECFCs praticamente pura colhida em densidade muito baixa chapeamento de 3 ou 10 células por centímetro quadrado em placas de cultura de células para permitir o crescimento única colônia. </li><li> Troca de um terço da média duas vezes por semana. </li><li> Depois de 14 dias (fim da cultura) remova o meio, lave duas vezes com PBS e corrigir as colônias com gelo solução de fixação a frio (acetona: metanol = 3, 2) por 15 minutos na geladeira. </li><li> Descartar a solução de fixação e deixar as placas aberto para secar por 10 minutos. </li><li> Hidratar as células com água destilada por 10 minutos. </li><li> Adicionar solução de hematoxilina de Harris e mancha as colônias fixadas por 12 minutos. </li><li> Remover H solução matoxylin e enxaguar as placas com água da torneira para se livrar da solução corante remanescente. </li><li> Tirar fotos de cada colônia porusando um estereomicroscópio e importação *. jpg ou *. tif formato em seu computador. </li><li> Abra as fotos com software ImageJ e analisar o número de células exata de cada colônia, conforme descrito na animação. </li><li> Abra o software ImageJ e importar uma imagem colônia usando o &#39;File \ Open &quot;função no menu suspenso. </li><li> Continuar usando o &quot;Processo de \ Background Subtrair&quot; função </li><li> Use a função &#39;seleção elíptica * * escova ou&#39; no menu ImageJ para marcar a margem colônia. </li><li> Limpar a imagem ao redor usando o &quot;Editar \ Fora Clear &#39;função e cortar a imagem, selecionando&#39; Imagem Cortar \ &#39;. </li><li> Ajustar o limite manualmente usando o &quot;Imagem \ Ajuste \ Threshold&quot; função, para que todas as células estão completamente coloridos em vermelho. </li><li> Conte o número de células da colônia, selecionando &quot;Analisar \ Analise Partículas &#39;. Escolha as configurações apropriadas (otimizadas para cada tipo de célula) e selecione &#39;OK&#39;. A caixa de resultados, incluindo contagem de células e uma nova imagem contendo os contornos correspondentes das células contadas aparecerá. <img src="/files/ftp_upload/1525/1525op2.jpg" alt="Figura 1" /></li></ol>

<ol>
	<li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+and+preclinical+production+of+human+lineage-negative+cells+for+regenerative+stem+cell+therapy.">Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy.</a> Transfusion. 45, 315-326 (2005).</li></ol>

Os autores não têm quaisquer conflitos financeiros de interesse a declarar.

Há uma variedade de fontes de onde começar ou MSCs ECFCs incluindo a medula óssea, tecido adiposo, sangue do cordão umbilical, etc É necessário ter ambos os tipos de progenitores da mesma fonte para comparar diretamente o envolvimento dos diferentes tipos de células em processos como tecidos e regeneração de navio ou de contribuição para a progressão do tumor. A facilidade com que a isolar e, posteriormente, estudar pares autólogo de ECFCs e MSCs do ...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>alpha modified MEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-GLutamin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EBM</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Single cytokine quots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75 cm2 flasks</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>150 cm2 plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell scraper</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin/EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monoclonal antibodies</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>500 mL filter 20 &mu;m pore size </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Preservative free heparin</td>
<td>&nbsp;</td>
<td>Biochrom</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (mscs) and endothelial colony forming progenitor cells (ecfcs)

O cordão umbilical é uma fonte rica de células progenitoras com alto potencial proliferativo, incluindo células mesenquimais estromais (também chamadas de células-tronco mesenquimais, MSCs) e endoteliais colônia formando células progenitoras (ECFCs). Ambos os tipos de células são atores-chave na manutenção da integridade dos tecidos e são, provavelmente, também envolvidos nos processos de regeneração e formação de tumor. Para estudar a sua biologia e funcionam de uma forma comparativa, é importante ter os dois tipos de células disponíveis a partir do mesmo doador. Também pode ser benéfico para fins regenerativos para derivar MSCs e ECFCs do mesmo tecido. Porque terapias celulares deve eventualmente encontrar seu caminho de laboratório para o leito que estabeleceu um novo método para isolar e ampliar ainda mais as células progenitoras sem o uso de proteína animal. Lisado de plaquetas agrupadas humano (pHPL) substituiu soro fetal bovino em todas as etapas do nosso protocolo para evitar completamente contacto das células com proteínas xenogénicos. Este vídeo demonstra uma metodologia para o isolamento e expansão de células progenitoras de um cordão umbilical. Todos os materiais e procedimentos serão descritos.

Watch this Scientific Journal Video about Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Este protocolo descreve o isolamento ea subsequente expansão de células mesenquimais estromais e as células endoteliais que formam colônias sem o uso de animais soro autólogo para gerar pares para fins de transplante experimental.

Isolamento e animais de Expansão de soro livre de cordão umbilical humano Derivado células mesenquimais estromais (MSCs) e endoteliais Células Progenitoras Colony Forming (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

isolation-animal-serum-free-expansion-human-umbilical-cord-derived

Research

JoVE Journal

Biology

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

15.4K visualizações.  Medical University of Graz, Austria. Este protocolo descreve o isolamento ea subsequente expansão de células mesenquimais estromais e as células endoteliais que formam colônias sem o uso de animais soro autólogo para gerar pares para fins de transplante experimental.

Vídeo: Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolamento de células endoteliais da veia umbilical humana (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biologia

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 ...

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

Isolamento e Enriquecimento de células-tronco mesenquimais Rat (MSC) e Separação de-colônia Único Derivado MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

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

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

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

Isolamento da Human Umbilical células musculares arteriais (HUASMC)

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

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Caracterização fenotípica e funcional das células endoteliais Formadoras de Colônias derivado do sangue do cordão umbilical humano

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

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

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

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

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

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation of Murine Valve Endothelial Cells

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

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

O isolamento de células endoteliais de Murino Válvula

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

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolamento de CD146<sup&gt; +</sup&gt; Células Residente Lung estromais mesenquimais de rato Pulmões

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Isolamento e enriquecimento de células progenitoras epiteliais pulmonares humanas para a cultura organoide

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...