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>Corning</td>
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			<td>Lonza</td>
			<td>CC-3151</td>
			<td></td>
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			<td height="21" style="height:21px;">Insulin</td>
			<td>Lonza</td>
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			<td>SingleQuots&#8482; Kit</td>
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			<td height="21" style="height:21px;width:216px;">EGF</td>
			<td>Lonza</td>
			<td style="width:119px;">CC-4136</td>
			<td>SingleQuots&#8482; Kit</td>
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			<td>Sigma</td>
			<td>E9644</td>
			<td>end concentration: 20 ng/ml</td>
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			<td>PeproTech</td>
			<td>100-18B</td>
			<td>end concentration: 20 ng/ml</td>
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			<td>Invitrogen/ Gibco</td>
			<td>17504-044</td>
			<td>end concentration: 1x</td>
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			<td height="21" style="height:21px;">Heparin-Natrium-25000 IE</td>
			<td>Ratiopharm</td>
			<td style="width:119px;">N68542.02</td>
			<td>dilution 1:1000</td>
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			<td>Gibco</td>
			<td style="width:119px;">15140-122</td>
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			<td>Gibco</td>
			<td style="width:119px;">10500-064</td>
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			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Gibco</td>
			<td style="width:119px;">21875-034</td>
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			<td height="21" style="height:21px;">Trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
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			<td height="21" style="height:21px;">Dulbecco&#8217;s PBS (1x)</td>
			<td>Gibco</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
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			<td height="21" style="height:21px;">Shield1&#160;</td>
			<td>Clontech</td>
			<td style="width:119px;">632189</td>
			<td>dilution 1:1000</td>
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			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td>Gibco</td>
			<td style="width:119px;">21041-025</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">DMEM/F12 (powder)</td>
			<td>Gibco</td>
			<td style="width:119px;">42400-010</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl cellulose</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">M0387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td>applichem</td>
			<td style="width:119px;">A2856</td>
			<td></td>
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			<td height="21" style="height:21px;">cell sorter&#160;</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>Aria III cell sorter&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS analyser</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>accuri c6 flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope</td>
			<td style="width:112px;">Olympus&#160;</td>
			<td style="width:119px;"></td>
			<td>IX50 Osiris</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 ...

evaluation-stem-cell-properties-human-ovarian-carcinoma-cells-using

Research

JoVE Journal

Medicine

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.

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Medicina

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Avaliação do Câncer de Migração Stem Cell Usando compartimentar dispositivos microfluídicos e imagens de células vivas

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Transplante de Células-tronco em um In vitro Modelo de isquemia / reperfusão Simulated

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Processamento de tecido primário do tumor cerebral para Ensaios de Células Tronco e Fluxo de Classificando

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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 ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

Baseado em celular Protocolo Ensaio para a predição prognóstica de escoliose idiopática Usando celular dielétrica Espectroscopia

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

RNAscope para<em&gt; Em situ</em&gt; Detecção de Papilomavírus Humano transcricionalmente ativo em Cabeça e Pescoço Carcinoma de células escamosas

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Multieletrodos Recordings Matriz de Human epiléptica pós-operatória Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Enriquecimento para resistente à quimioterapia do cancro do ovário células-tronco de linhas de células humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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 ...