This study was supported by a grant from the Baden-W&#252;rttemberg Stiftung (Adult Stem Cells Program II) awarded to C.L. We thank Dr. Martina Konantz for critical input and review of the manuscript. We thank Emmanuel Traunecker and Toni Krebs from the DBM FACS Facility (University Hospital Basel) for assistance with FACS sorting.

1. Geração de células humanas de carcinoma de ovário OVCAR-3 estavelmente transduzidas com Lentiviruses Contendo o Reporter Construct SOX2 Região Regulatory <ol><li> Gerar partículas lentivirais por transfecção da linha celular 293T HEK-embalagem com uma construção repórter reconhecer uma região reguladora SOX2 como descrito 10,21. NOTA: O repórter construir contém ainda um domínio desestabilização do System Shield ProteoTuner à frente da proteína tdTomato fluorescência. Shield1 se liga ao domínio desestabilização impedindo assim o proteassoma para degradar a proteína de fluorescência 22. </li><li> Transduzir células OVCAR-3 com partículas lentivirais durante um período de tempo de 24 horas. Em seguida, remover o sobrenadante viral e lavar as células com solução salina de fosfato tamponada (PBS) e cultivadas em meio completo (meio RPMI suplementado com 10% de FBS, 100 U / ml de penicilina, 100 ug / ml de estreptomicina). </li><li> 48 horas depois, 10 ug / ml puromicina foram adicionados às culturas e mantidas durante 5 dias para permitir a selecção de células transduzidas adequadamente. </li></ol> 2. Preparação de células de classificação e Galvanização <ol><li> Adicionar Shield1 a 1: 1000 de diluição 24 h antes da separação de células. Use estavelmente transduzidas OVCAR-3 em células sem tratamento Shield1 como controlos negativos (Figura 1). Aspirar meios de balão, lavar as células com PBS 1x e trypsinize células com 0,05% Tripsina-EDTA durante 3 min. </li><li> Pare tripsina utilizando meio completo (ver acima), contar o número de células, as células de centrifugação a 300 xg à temperatura ambiente (15 - 25 ° C) durante 5 min. </li><li> Decantar o sobrenadante e ressuspender as células cuidadosamente em 0,5-1 ml de PBS estéril. </li><li> Use 40 um filtro cap filtro de células para obter a suspensão de uma única célula. </li><li> Ajustar a contagem de células a 5 milhões de células por ml. </li><li> Prepare baixa fixação placas ultra-96 poços com 100 ul esferas médio (MEGM suplementado com fatores de crescimento, citocinas e suplementos,B-27, heparina-sódio; ou DMEM / F12 suplementado com factores de crescimento, citocinas e suplementos, B-27, heparina-sódio, com ou sem adição de 1% de metilcelulose, ver também a Tabela 1). Opcionalmente adicionar antibióticos para o meio a uma concentração de 100 U / ml de penicilina e 100 ug / ml de estreptomicina para minimizar o risco de possível contaminação. </li><li> Ordenar as células em RFP- preparadas placas de 96 poços a partir de cima e RFP +, uma célula por cavidade (ensaio de esferas único baseado em células), e 100 células por poço (de ensaio de multi esferas baseados em células), respectivamente. Execute classificador de células tipo em comercialmente disponível (ver Materiais) usando o modo de única célula, Sort setup: 100 μ bocal, pressão do invólucro 20 psi, e rendimento de máscara 0, pureza máscara de 32, a máscara de fase 16. </li><li> Avaliar a eficiência do plaqueamento por microscopicamente conseguir poços contendo células (para o ensaio de base celular único) e por contagem do número de células em poços individuais (para o ensaio baseado em células múltiplas; Figura 2 </strong&gt;). </li><li> Incubar as células em condições normais médias em esferas (para a composição ver passo 2.6) a 37 ° C e 5% de CO 2. Suplemento de bFGF por dia (20 ng / ml) e EGF (20 ng / ml). </li><li> Após uma semana, contar o número de esferas de tumor emergente usando um microscópio padrão com 4X ou ampliação de 10X e um microscópio de fluorescência para detectar o sinal de fluorescência a partir do sistema repórter integrada. Contagem de esferas com um diâmetro superior a 100 um, como esferas &quot;grandes&quot; e as esferas com um diâmetro de 50 - 100 mm como &quot;pequenos&quot; esferas (Figura 3). Tenha certeza de que você contar esferas reais e não celular clusters. NOTA: Em ensaios baseados em células individuais formação esfera é mais fácil para marcar microscopicamente após 10 (contra 7) dias de cultura. </li><li> Calcular a proporção de células formadoras de esfera em RFP + e células, respectivamente RFP- em ensaios baseados em células individuais (uma placa de 96 poços para cada experiência individual) ou de multi-célula baesferas ensaios sed (um bem para cada experimento indivíduo), como apresentado por Shaw et al. 16 NOTA: Proporção de células formando esfera (%) = (número de esferas) / (número de células semeadas) x 100 </li></ol> 3. Passaging Serial de Esferas <ol><li> Colocar o conteúdo de cada cavidade dentro de um tubo estéril adequado e centrifugar a 300 xg durante 10 min à temperatura ambiente. Para baseados em células múltiplas esferas ensaios, reúna as esferas de um bem. Lava-se a bem 3 - 5 vezes com PBS e centrifugação mais 2 min. Para individuais baseados em células esferas ensaios, coletar esferas individuais. Devido ao baixo número de células, utilizar tubos de 1,5 ml para centrifugação e lavagem passos. </li><li> Remover o sobrenadante e ressuspender o sedimento em 200 ul de 0,05% de tripsina-EDTA. </li><li> A fim de conseguir a separação celular óptimo, incubar a suspensão de células a 37 ° C durante 5 min num agitador suave. Otimizar tripsinizaï¿½o tempo para a sua linha de células de menor taxa de morte celular: se nenhuma grandeesferas é triturado visível delicadamente com uma pipeta de ponta de 100 mL. No caso de grandes esferas ainda estão presentes, incube com tripsina outro 3 min e, em seguida, avançar para a etapa de trituração. No caso de um único ensaios baseados em células para se certificar de optimizar o tempo de rendimento óptimo das células de células vivas durante o passo de tripsinização. </li><li> Para inactivar a tripsina, adicionar 500 ul meio completo e centrifugação a 300 xg durante 10 min, no caso de ensaios de células individuais, centrifugar durante mais 2 min. </li><li> Remover sobrenadante e ressuspender as células em meio de esferas cuidadosamente. </li><li> Usar um filtro de 40 um filtro de células de tampão para se obter uma suspensão de célula única. </li><li> No caso de se utilizar células RFP- em ensaios bassed de células multi-RFP + e, avaliar a percentagem de células fluorescentes em cada poço após ressuspensão através de análise de citometria de fluxo. </li><li> Para os ensaios de replating série de células individuais, sementes de 1 célula por cavidade em um novo ultra-baixo fixação placa de 96 poços preparados tal como acima descrito. A partir de uma esfera individual, semear aproximadamente 20 poços individuais. Para os ensaios de esferas replating primários múltiplos baseados em células, as células de semente obtido a partir de um poço de esferas primárias para uma nova placa de 96 poços e contar o número de células no dia seguinte por microscopia. </li><li> Avaliar a percentagem de células formadoras de esfera em esferas secundárias ensaios usando a fórmula descrita no passo 2.11. </li></ol> Análise 4. Resultado <ol><li> Analisar os resultados de experimentos realizados em triplicatas independentes e usar o teste t de Student dois lados para analisar os valores normalmente distribuídos e de outra forma Mann-Whitney-testes para análise estatística. </li></ol>

<ol>
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The authors have nothing to disclose.

Esferas culturas é um método amplamente utilizado para testar o potencial de células estaminais do cancro e para enriquecer as células estaminais do tipo em uma ampla variedade de células tumorais humanas 15,25,26. Sob estas condições de cultura, as células cancerosas que não têm capacidade de auto-renovação são esperados para diferenciar e eventualmente sofrem morte celular. Embora eles podem, inicialmente, formar grupos de células tumorais ou mesmo esferas especialmente em ensaios preliminares...

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.
A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehyde dehydrogenase (ALDH) activity4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable6. Consistently, in ovarian cancer, other than for example in breast carcinoma7, expression of ALDH1 associates with favorable outcome8 and expression of the proposed stem cell marker CD44 variant has no prognostic value9. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells10 and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.
By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice10,13.
An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells14. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast17, brain18, pancreas19 and colon20 tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models10. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells10. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo10 suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.
In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

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			<td>accuri c6 flow cytometer</td>
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evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

Em ensaios de esferas convencionais, cerca de 40% de RFP + OVCAR-3 células vs. 20% de células RFP- deu origem a uma esfera tumor individual no ensaio de esferas primário (Figura 4A). Além disso, as esferas formadas por RFP + células eram maiores em tamanho do que os que são formados por células RFP-. Quando colocadas em ensaios baseados em células individuais, células RFP + também formou mais esferas do que as células RFP-, confirmando os resultados acima. No enta...

Watch this Scientific Journal Video about Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays at JoVE.com

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Avaliação das Propriedades Stem Cell em carcinomas de células do ovário humano utilizar multi e individuais baseados em células Esferas Ensaios

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

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9.5K visualizações.  University Hospital Basel. In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.