Este trabalho foi financiado pelo Centro de Pesquisa da Flórida tumor cerebral, o Preston A. Wells Jr. Centro de Terapia do tumor cerebral e os Institutos Nacionais de Saúde (1R21CA141020-01).

1. Preparando Glioblastoma suspensão de célula única <ol><li> Cultura Gliomasphere é estabelecido e mantido utilizando o ensaio neuroesfera (NSA), como descrito anteriormente 2,11,12. </li><li> No momento apropriado para as passagens em gliomaspheres, o meio contendo as esferas é removido, colocado num tubo de cultura apropriado tamanho estéril de tecidos, e centrifugadas a 800 rpm (110 g) durante 5 min à temperatura ambiente. </li><li> O sobrenadante é eliminado eo sedimento de esferas é ressuspenso em 1 ml de 0,05% de tripsina-EDTA e incubado a 37 ° C num banho de água durante 3-5 min. </li><li> Um volume igual de inibidor de tripsina de soja é, então, utilizado para parar a actividade de tripsina. </li><li> A suspensão celular é suavemente pipetados para cima e para baixo, para assegurar a homogeneidade e a neutralização completa da actividade da tripsina. </li><li> A suspensão de células é novamente centrifugado a 800 rpm durante 5 min. O sobrenadante é removido e as células são ressuspensas em 1 ml de NeuroCultNSC Meio Basal. </li><li> 10 uL da suspensão de célula única é misturado com 90μl de azul de tripano para realizar uma contagem de células. </li></ol> 2. A coloração com celular Rastreamento Carboxifluoresceína Succinimidyl Ester (CFSE) Verde corante fluorescente <ol><li> Por cada 1 milhão de células a ser corado, 1 ml do reagente de coloração é preparado pela adição de 1 ul de 5 mM de corante CFSE celular Trace (Molecular Probes, Invitrogen) a 1 ml de Meio NeuroCult NSC Basal para dar uma concentração final de coloração com 5 uM . </li><li> O reagente de coloração que contém as células é bem misturada até à homogeneidade e incubados à temperatura ambiente durante 10 min. </li><li> Durante a incubação, gelada NeuroCult Meio Basal NSC é preparada como a solução de têmpera. </li><li> Para interromper o processo de coloração, aproximadamente 5-10 volumes de solução gelada de extinção é adicionado às células CFSE-carregados. </li><li> A solução é, então, centrifugadas a 800 rpm durante 5 min e o sobrenadante é descartado. </li><li> Ao ªÉ ponto, deve aparecer um pellet de células na parte inferior do tubo com um matiz brilhante de verde de cor, indicando a coloração foi bem sucedida. </li><li> As células são lavadas por ressuspensão do sedimento em 1-2 ml de meio fresco NSC NeuroCult Basal, centrifugação a 800 rpm durante 5 min e descartando o sobrenadante. Este procedimento é repetido para um total de três lavagens. </li><li> Após a terceira lavagem, as células são ressuspensas em 1 ml de NeuroCult Meio Basal NSC e uma contagem de células é realizada. </li><li> As células CFSE coradas são então semeadas em frascos de cultura de tecidos estéreis, a uma densidade adequada [50000 células / ml] e colocados em uma de 37 ° C / 5% de CO2 incubadora humidificada. </li></ol> 3. Verificação Carregando CFSE <ol><li> As células plaqueadas em cultura NSA pode ser monitorizado sob um microscópio de fluorescência para verificar a coloração CFSE. Nesta fase, todas as células exibem cor verde brilhante (Figura 1). Alternativamente, uma gota da suspensão de células a partir deCFSE células coradas pode ser colocado sobre uma lâmina de microscópio e coberta com uma lamela para visualizar as células sob um microscópio fluorescente. </li><li> Rotulagem CFSE sucesso é ainda confirmada por meio de citometria de fluxo. Tal como muitos como 1-5 x 10 5 células de cada grupo são suficientes para a verificação de coloração CFSE. O CFSE células carregadas e descarregadas células de controlo negativo, respectivamente, são de novo suspensas em 1 x PBS-ou NeuroCult Meio Basal NSC para um volume total de 500 ul e colocados em tubos separados FACS. </li><li> O citômetro de fluxo é ajustada com base em suas instruções manual do usuário para os parâmetros relacionados. CFSE tem um máximo de comprimento de onda de excitação de 492 nm e um máximo de emissão de 517 nm. </li><li> Em primeiro lugar, as células de controlo não carregados são executados e os eventos adquiridos são registadas em dispersão frontal e lateral (FSC vs SSC) e histograma, e a população de células principal está fechado (Figura 2A). </li><li> Do mesmo modo, as células CFSE carregados são analisados ​​usando o mesmo parâmetros utilizados para as células de controlo (Figura 2B). </li></ol> 4. O isolamento do Slow-divisão [CFSE ie Retenção] População de células utilizando separação de células activada fluorescente (FACS) <ol><li> Após a divisão, a intensidade é reduzida e CFSE células hGBM formar gliomaspheres que são compostos de células que exibem diferentes níveis de intensidade de fluorescência verde (Figura 3). Quando os gliomaspheres ter atingido um tamanho médio de cerca de 150-200 um de diâmetro (cerca de 5 a 10 dias pós-carregamento CFSE dependendo da taxa de crescimento da linha de células), o meio contendo as esferas são removidas, colocadas em cultura de tecido apropriado um tamanho estéril tubo, e centrifugadas a 800 rpm durante 5 min à temperatura ambiente. </li><li> A suspensão de célula única é preparado como descrito nas etapas anteriores e as células são ressuspensas em PBS para uma contagem de células, a fim de preparar uma suspensão de células com uma densidade de até 10 7 células por mililitro. </li><li> À réer ressuspensão das células num volume apropriado de PBS, iodeto de propídio (PI, Sigma, 500 ug / ml) é adicionado a uma concentração de 1 ul / ml de suspensão de células para excluir as células mortas quando ordenadas / analisados ​​por citometria de fluxo. </li><li> Dois tubos de 15 ml de tecido estéreis cultura de cada 1 ml contendo de completa Médio NSC NeuroCult é usado para coletar as células ordenadas lento divisórias (células CFSE de retenção) e as células de divisão rápida (células CFSE diluição). </li><li> Em primeiro lugar, a suspensão de células a partir do grupo de células descarregado é executado através do citómetro de fluxo. </li><li> As células (eventos) são traçados com base em dispersão frontal (FSC) versus dispersão lateral (SSC) e parâmetros de tensão de ambos os parâmetros são ajustados de modo que a maioria das células são incluídos no fluxo de trama. </li><li> Para excluir as células mortas ou danificadas, as células são traçados com base na reactividade contra SSC PI ea população única célula viva (PI negativo) é fechado. </li><li> Em seguida, uma população de células homogéneas é seleccionado com base na fdispersão orward (FSC) e lado de dispersão (SSC). </li><li> A população de células homogéneas único vivo é em seguida representados num histograma com base na frequência de células versus intensidade de CFSE definido em uma escala logarítmica. A intensidade do laser CFSE é ajustada por forma a colocar o sinal negativo entre 0 e 100. </li><li> Utilizando os mesmos parâmetros e uma estratégia de gating semelhante, as células CFSE rotulados são executados por meio da citometria de fluxo e analisadas (Figura 4C). </li><li> Subsequentemente, uma população de células de divisão lenta e uma população total (células que se dividem mais rapidamente) são identificados com base na sua capacidade de reter CFSE (CFSE alta superior a 5% e inferior CFSE baixa de 85%, respectivamente). </li><li> Estas populações de células diferentes são então classificadas em diferentes tubos de 15 ml de tecidos estéreis de cultura contendo 1 mL de meio completo NSC. </li><li> Células seleccionadas são então centrifugadas a 800 rpm durante 5 min. </li><li> O sobrenadante é eliminado eo sedimento é ressuspenso em um adeo volume adequado de meio (dependendo do número de eventos isolados ou tamanho pellet) e uma contagem de células é realizada. </li><li> As células a partir das diferentes populações classificadas são então colocadas em placas a 50.000 células por mililitro, em um frasco de cultura de tecido estéril apropriado e colocadas numa 37 ° C / 5% de CO2 incubadora humidificada durante 5-10 dias, antes de ser passado seriadamente ou utilizados em experiências a jusante. </li></ol> 5. Resultados representativos Após o carregamento CFSE, todas as células apresentam uma fluorescência verde intensa como visualizada ao microscópio (Figura 1). Esta cor esverdeada pode ser visto até mesmo a olho nu (veja o vídeo). Analisando estas células por citometria de fluxo também mostra muito intensa fluorescência verde em comparação com células de controlo (Figura 2). Após células em placas de cultura CFSE carregados neuroesfera, estas células proliferam e formam gliomaspheres 150-200 uM em 5-10 dias. À medida que as células proliferam, t<html> ele corante fluorescente é distribuído igualmente entre as células filhas após divisões, conduzindo à redução para metade da intensidade de fluorescência em cada divisão celular. Glioblastomas são funcionalmente heterogéneas e são compostos de células que proliferam em diferentes escalas de tempo. O método descrito no presente protocolo permite identificar e isolar rápida e lenta de células em divisão, em virtude da sua capacidade de diluir ou reter CFSE, respectivamente (Figura 3, ver também o vídeo). A análise de citometria de fluxo de gliomaspheres dissociadas gerados a partir de CFSE células carregadas, em comparação com células não carregados demonstra as propriedades de retenção de CFSE diversas (Figura 4). Células em divisão rápida (85% inferior) ou lentas as células em divisão (a 5% do topo - CFSE alta) gliomapshere exibem capacidade de formação, quando cultivadas nas condições do ensaio neuroesfera (Figura 5). gura 1 &quot;/&gt; Figura 1. Células tumorais dissociadas imediatamente após carregamento com corante CFSE. As células apresentam cor verde brilhante demonstrando rotulagem sucesso. Ampliação original, 10 x. <img src="/files/ftp_upload/3918/3918fig2.jpg" alt="Figura 2" /> Figura 2. Confirmação CFSE carregamento utilizando citometria de fluxo. A) CFSE células descarregado, B) carregados células e c) comparação da intensidade de fluorescência em células CFSE carregados contra descarregado. Note-se que há várias ordens de magnitude diferença na intensidade de fluorescência entre as células marcadas e não marcadas. <img src="/files/ftp_upload/3918/3918fig3.jpg" alt="Figura 3" /> Figura 3. Um representante gliomasphere derivada de uma célula CFSE-loaded 6 dias após o plaqueamento. Algumas células apresentam coloração CFSE muito brilhante. Ampliação original; 63 x. <img src = &quot;/ files/ftp_upload/3918/3918fig4.jpg&quot; alt = &quot;Figura 4&quot; /&gt; Figura 4. Representante de citometria de fluxo / parcelas histogramas para o isolamento rápido e lento de células em divisão 6 dias após coloração com CFSE. A) FSC vs SSC para mostrar a população de célula inteira, B) versus reactividade SSC PI para excluir as células mortas ou danificadas, C) FSC vs SSC a porta de uma população homogénea de células vivas, D) demonstra a presença de células CFSE retenção 6 dias após a rotulagem CFSE como evidenciado por uma maior intensidade de fluorescência em comparação com o controlo negativo. O histograma mostra que a intensidade CFSE deteriorado ao longo do tempo. E) Histograma mostrando a estratégia modular para identificar as células de fixação (CFSE top 5%) e as células CFSE diluição inferior (85%). <img src="/files/ftp_upload/3918/3918fig5.jpg" alt="Figura 5" /> Figura 5. Gliomaspheres representativos gerados a partir de <stronG&gt; A) fast-divisão de células CFSE (baixo) e células B) slow-divisão (CFSE alta). Ampliação original:. 10x e 20x, respectivamente C) contagem de células Representante obtidas rápido ou lento dividindo cultura de células derivadas no tempo de passagem. P &lt;0,05, t-teste. <a href="http://www.jove.com/files/ftp_upload/3918/3918fig5large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

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Um crescente número de estudos demonstraram a importância de uma lenta dividindo compartimento sub-células de cancro em que contribuem para a formação de tumores e resistência ao tratamento 2-8. Por isso, é crítico para descrever e padronizar os métodos experimentais que permitem o estudo desta subpopulação específica de células ou, mais geralmente para comparar as subpopulações com diferentes taxas de crescimento. Usando CFSE para identificar e isolar sub-fracções de células com base na sua...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Tipo </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> NeuroCult Médio NSC Basal (Humana) </td><td> Médio </td><td> Stem Cell Technologies </td><td> 05750 </td></tr><tr><td> NeuroCult NSC Suplementos Proliferação (Humana) </td><td> Meio suplementado </td><td> Stem Cell Technologies </td><td> 05753 </td></tr><tr><td> 0,05% de tripsina-EDTA </td><td> Reagente </td><td> Gibco </td><td> 25300-062 </td></tr><tr><td> Inibidor de tripsina de soja </td><td> Reagente </td><td> Sigma </td><td> T6522 </td></tr><tr><td> Penicilina / estreptomicina </td><td> Reagente </td><td> Gibco </td><td> 15140-122 </td></tr><tr><td> T25 frasco </td><td> Cultura de conservação </td><td> Nalge Nunc internacional </td><td> 136196 </td></tr><tr><td> T80 frasco </td><td> Cultura de conservação </td><td> Nalge Nunc internacional </td><td> 178905 </td></tr><tr><td> Tubos de 15 ml </td><td> Cultura de conservação </td><td> BD Falcon </td><td> 352096 </td></tr><tr><td> Tubos de 50 ml </td><td> Cultura de conservação </td><td> BD Falcon </td><td> 352070 </td></tr><tr><td> EGF </td><td> Fator de crescimento </td><td> R &amp; D </td><td> 2028-EG </td></tr><tr><td> CFSE CellTrace Kit Proliferação de Células </td><td> Divisão Celular fluorescente Rastreamento Dye </td><td> Molecular Probes, Invitrogen </td><td> C34554 </td></tr></tbody></table>

identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (cfse)

Heterogeneidade tumoral representa uma característica fundamental apoio robustez tumor e apresenta um obstáculo fundamental para o desenvolvimento de estratégias terapêuticas 1. Para superar o problema da heterogeneidade de tumores, que é essencial para desenvolver ensaios e ferramentas que permitam fenotípica, identificação (epi) genéticos e funcionais e caracterização de sub-populações de tumor que conduzem a doenças específicas e patologias representam alvos clinicamente relevantes. Está agora bem estabelecido que os tumores apresentam distintas sub-fracções de células com diferentes frequências de divisão celular, e que os critérios funcionais de ser lento ciclismo está positivamente associado com a capacidade de formação de tumor em vários cancros, incluindo as do cérebro, da mama, pele e pâncreas, bem como leucemia 2-8. O corante fluorescente carboxifluoresceína succinimidil éster (CFSE) tem sido utilizado para controlar a frequência de divisão de células, in vitro e in vivo em sangue-carregada tumors e tumores sólidos tais como o glioblastoma 2,7,8. A célula-permeante não fluorescente pró-droga é convertido de CFSE por esterases intracelulares para um composto fluorescente, a qual é retida no interior das células por ligação covalente a proteínas através da reacção da sua porção de succinimidilo com grupos amina intracelulares para formar ligações amida estáveis ​​9. O corante fluorescente é distribuído igualmente entre as células filhas após divisões, conduzindo à redução para metade da intensidade de fluorescência em cada divisão celular. Isso permite o acompanhamento da freqüência do ciclo celular até 8-10 rodadas de divisão 10. Capacidade de retenção de CFSE foi utilizado com células tumorais do cérebro para identificar e isolar uma subpopulação de ciclismo lenta (topo 5% de retenção de corante de células) demonstraram a ser enriquecida em actividade de células do cancro da haste 2. Este protocolo descreve a técnica de coloração de células com CFSE e o isolamento de populações individuais dentro de uma cultura de GLIO humanoblastoma (GBM) derivadas de células que possuem taxas de divisão diferentes usando citometria de fluxo 2. A técnica tem servido para identificar e isolar uma população de tumor cerebral ciclismo lenta das células por virtude da sua capacidade para reter a rotulagem CFSE.

Watch this Scientific Journal Video about Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE at JoVE.com

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

Este protocolo de vídeo demonstra a aplicação do corante fluorescente carboxifluoresceína succinimidil éster (CFSE) para a identificação e separação de diferentes sub-populações de células em glioblastoma humano com base na frequência de divisão celular.

Identificação e isolamento de células em divisão lento no glioblastoma humano usando Carboxifluoresceína Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

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JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

14.0K visualizações.  The University of Florida. Este protocolo de vídeo demonstra a aplicação do corante fluorescente carboxifluoresceína succinimidil éster (CFSE) para a identificação e separação de diferentes sub-populações de células em glioblastoma humano com base na frequência de divisão celular.

Vídeo: Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

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

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Geração de Microtumors Usando 3D Biogel Humano Sistema de Cultura e células de glioblastoma derivado do paciente para Kinomic Profiling e Testes resposta à droga

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

Entrega intranasal de células-tronco terapêuticas para Glioblastoma em um modelo de mouse

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Caracterização de células imunes no tecido adiposo humano usando citometria de fluxo

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolamento de células humanas primárias do Decidual de membranas fetais de termo placentas

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...