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. Génération des droits de cellules de carcinome de l&#39;ovaire de OVCAR-3 transduites de manière stable avec lentivirus contenant le rapporteur Construct région régulatrice SOX2 <ol><li> Générer des particules lentiviraux par transfection de la lignée cellulaire HEK 293T-emballage avec une construction de rapporteur de reconnaître une région de régulation de la manière décrite SOX2 10,21. REMARQUE: La construction de rapporteur contient en outre un domaine de déstabilisation du système ProteoTuner bouclier avant de la protéine de fluorescence tdTomato. Shield1 se lie au domaine de déstabilisation empêchant ainsi le protéasome de dégrader la protéine de fluorescence 22. </li><li> Transduire des cellules OVCAR-3 avec des particules lentivirales sur une période de temps de 24 heures. Par la suite, retirer le surnageant viral et laver les cellules avec une solution saline tamponnée phosphate (PBS) et mises en culture dans du milieu complet (RPMI complété avec 10% de FBS, 100 U / ml de pénicilline, 100 ug / ml de streptomycine). </li><li> 48 h plus tard, 10 ng / ml puromycine a été ajouté aux cultures et maintenue pendant 5 jours pour permettre la sélection de cellules transduites correctement. </li></ol> 2. Préparation de la cellule de tri et de Placage <ol><li> Ajouter Shield1 au 1: 1000 dilution 24 heures avant le tri de cellules. Utilisation transduites de manière stable des cellules OVCAR-3 sans traitement Shield1 comme témoins négatifs (Figure 1). Aspirer le milieu de flacon, laver les cellules avec PBS 1x et Trypsiniser cellules avec 0,05% de trypsine-EDTA pendant 3 min. </li><li> Arrêtez la trypsine en utilisant un milieu complet (voir ci-dessus), compter le nombre de cellules, les cellules de centrifugation à 300 xg à température ambiante (15-25 ° C) pendant 5 min. </li><li> Décanter le surnageant et remettre les cellules soigneusement 0,5 à 1 ml de PBS stérile. </li><li> Utilisez 40 um filtre de bouchon de crépine de cellules pour obtenir une suspension à cellule unique. </li><li> Ajuster le nombre de cellules de 5 millions de cellules par ml. </li><li> Préparer faible fixation des plaques à 96 puits ultra avec 100 ul de milieu sphères (MEGM supplémenté avec des facteurs de croissance, les cytokines, et les suppléments,B-27, l&#39;héparine de sodium; ou DMEM / F12 supplémenté en facteurs de croissance, cytokines, et des suppléments, B-27, l&#39;héparine de sodium avec ou sans addition de 1% de méthylcellulose, voir également le tableau 1). Ajouter éventuellement des antibiotiques dans le milieu à une concentration de 100 U / ml de pénicilline et 100 ug / ml de streptomycine afin de minimiser le risque de contamination possible. </li><li> Trier DP + et les cellules RFP- préparés dans des plaques à 96 puits à partir de ci-dessus, une cellule par puits (single sphères de dosage à base de cellules) et 100 cellules par puits de dosage (multi sphères à base de cellules), respectivement. Effectuer tri sur disponible dans le commerce trieur de cellules (voir Matériaux) en utilisant le mode unique de cellules, Trier configuration: 100 μ buse, la pression de la gaine 20 psi, et le masque de rendement 0, masque pureté 32, masque de phase 16. </li><li> Évaluer l&#39;efficacité de placage en marquant au microscope puits contenant des cellules (pour le dosage à base de cellules unique) et en comptant le nombre de cellules dans des puits individuels (pour le dosage à base de cellules multiples; Figure 2 </strong&gt;). </li><li> Incuber les cellules dans des conditions normales dans les sphères moyennes (pour la composition voir l&#39;étape 2.6) à 37 ° C et 5% de CO 2. Supplément quotidien bFGF (20 ng / ml) et de l&#39;EGF (20 ng / ml). </li><li> Après une semaine, compter le nombre de nouvelles sphères tumorales en utilisant un microscope standard avec un grossissement de 10X ou 4X et un microscope à fluorescence pour détecter le signal de fluorescence à partir du système rapporteur intégré. Comptez sphères avec un diamètre supérieur à 100 um comme «grands» des sphères et des sphères d&#39;un diamètre de 50 à 100 um comme &quot;petits&quot; domaines (Figure 3). Assurez-vous que vous comptez sphères réelles et non cellulaires grappes. NOTE: Dans les essais à base de cellules simples formation sphère est plus facile de marquer au microscope après 10 (contre 7) jours de culture. </li><li> Calculer la proportion de cellules de la sphère formation en DP + et les cellules respectivement RFP- dans des dosages à base de cellules simples (une plaque de 96 puits pour chaque expérience individuelle) ou plusieurs cellules-basphères sed essais (un puits pour chaque expérience individuelle) présentés par Shaw et al. 16 REMARQUE: Proportion de cellules formant sphère (en%) = (nombre de sphères) / (nombre de cellules ensemencées) x 100 </li></ol> 3. Le passage en série des Sphères <ol><li> Placez le contenu de chaque puits dans un tube et centrifuger stérile appropriée à 300 g pendant 10 min à température ambiante. Pour plusieurs sphères dosages à base de cellules, rassembler les sphères d&#39;un puits. Laver le bien 3-5 fois avec du PBS et centrifuger 2 min plus. Pour sphères simples tests à base de cellules, recueillir des sphères individuelles. En raison du faible nombre de cellules, utilisez tubes de 1,5 ml pour centrifugation et lavage étapes. </li><li> Retirer le surnageant et remettre en suspension le culot dans 200 pl de 0,05% de trypsine-EDTA. </li><li> Afin d&#39;obtenir une séparation optimale des cellules, incuber la suspension de cellules à 37 ° C pendant 5 min dans un agitateur doux. Optimiser trypsinisation temps pour votre lignée cellulaire à taux de mort cellulaire inférieure: si aucune grandesphères est trituré visible doucement à l&#39;aide d&#39;une pipette de 100 pi. En cas de grosses sphères sont toujours présents, incuber avec de la trypsine autre 3 min, puis passez à l&#39;étape de trituration. En cas de simple tests cellulaires veillez à optimiser le temps pour le rendement cellulaire optimal de cellules vivantes au cours de l&#39;étape de traitement à la trypsine. </li><li> Pour inactiver la trypsine, ajouter 500 pi de milieu complet et centrifuger à 300 g pendant 10 min, dans le cas de dosages cellulaires simples, centrifuger pendant 2 min supplémentaires. </li><li> Éliminer les cellules surnageant et remettre soigneusement dans le milieu des sphères. </li><li> Utiliser un filtre de plafonnement des tamis cellulaire de 40 um pour obtenir une suspension monocellulaire. </li><li> Dans le cas de l&#39;utilisation DP + et les cellules RFP- dans des dosages cellulaires bassed multiples, évaluer le pourcentage de cellules présentant une fluorescence dans chaque puits après remise en suspension par analyse cytomètre de flux. </li><li> Pour les essais de réensemencement série de cellules individuelles, semences 1 cellule par puits dans une nouvelle plaque de fixation basse ultra 96 ​​puits préparés comme détaillé ci-dessus. D&#39;une sphère individuelle, ensemencer environ 20 puits individuels. Pour les essais de réensemencement de multiples sphères primaires à base de cellules, les cellules obtenues à partir de graines et une des sphères primaires dans une nouvelle plaque à 96 puits et compter le nombre de cellules par microscopie lendemain. </li><li> Évaluer la proportion de cellules de la sphère formant dans les sphères secondaires essais utilisant la formule décrite à l&#39;étape 2.11. </li></ol> 4. Analyse des résultats <ol><li> Analyser les résultats des expériences réalisées en triple indépendants et utiliser le test t de Student recto-verso pour analyser les valeurs normalement distribués et autrement Mann-Whitney-Tests pour l&#39;analyse statistique. </li></ol>

<ol>
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The authors have nothing to disclose.

Sphères cultures sont une méthode largement utilisée pour analyser le potentiel de cellules souches du cancer et enrichir en cellules souches-comme dans un large éventail de cellules tumorales humaines 15,25,26. Dans ces conditions de culture, les cellules cancéreuses qui ne ont pas la capacité d&#39;auto-renouvellement se attend à différencier et, éventuellement, subir une mort cellulaire. Bien qu&#39;ils puissent former initialement des agrégats de cellules ou même des sphères de tumeurs primair...

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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			<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.

Dans les sphères des analyses classiques, près de 40% des DP + OVCAR-3 cellules contre 20% des cellules RFP- a donné lieu à une sphère de tumeur individuelle dans le dosage des sphères primaire (figure 4A). En outre, les sphères formées par DP + cellules sont plus grandes en taille que ceux qui sont formés par des cellules RFP-. Lorsque plaqués dans des essais à base de cellules simples, les cellules de la DP + également formés plusieurs sphères que les cellule...

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.

Evaluation des propriétés de cellules souches en cellules humaines de l&#39;ovaire Carcinome utilisant MULTI et simples à base de cellules Sphères dosages

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 vues.  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.