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.

安定SOX2規制地域レポーター構築物を含むレンチウイルスで形質導入されたOVCAR-3ヒト卵巣癌細胞の1世代<ol><li> 10,21が説明されているようにSOX2調節領域を認識レポーター構築とHEK 293T-パッケージング細胞株をトランスフェクションすることによってレンチウイルス粒子を生成します。 注：レポーター構築物はさらに、先にtdTomatoの蛍光タンパク質のProteoTunerシステムシールドシステムの不安定化ドメインを含む。 Shield1を、それにより蛍光タンパク質22を分解するためのプロテアソームを防止不安定化ドメインに結合する。 </li><li> 24時間の期間にわたって、レンチウイルス粒子を有するOVCAR-3細胞を形質導入する。その後、ウイル​​ス上清を除去し、リン酸緩衝生理食塩水（PBS）、完全培地中で培養した（RPMI、10％FBS、100 U / mlのペニシリン、100μg/ mlのストレプトマイシン）で細胞を洗浄する。 </li><li> 48時間後、10μg/ mlののプーロマイシンを培養物に添加し、適切に形質導入​​された細胞の選択を可能にするために5日間維持した。 </li></ol>細胞選別、めっきの作製<ol><li>前細胞選別1,000希釈24時間：1でのShield1を追加します。ネガティブコントロールとしてのShield1処理（ 図1）することなく、安定的に形質導入されたOVCAR-3細胞を使用してください。フラスコから培地を吸引除去し、3分間、0.05％トリプシン-EDTAを用いて細胞を1×PBSで細胞を洗浄し、トリプシン処理。 </li><li> 5分間 - （25°C 15）完全培地を使用してトリプシンを停止し（上記参照）、細胞数をカウントし、室温で300×gで遠心分離細胞。 </li><li> 1mlの滅菌PBS - 0.5に慎重に上清を再懸濁細胞をデカントします。 </li><li>単一細胞懸濁液を得るために、40μmのセルストレーナーキャップフィルタを使用する。 </li><li>ミリリットルあたり5万個の細胞に細胞数を調整します。 </li><li>培地を100μlの球で超低付着の96ウェルプレートを準備し（MEGMは、増殖因子、サイトカイン、およびサプリメントを補充B-27、ヘパリンナトリウム;またはDMEM / F12）は、 表1を参照して、1％メチルセルロースの添加の有無にかかわらず、成長因子、サイトカイン、およびサプリメント、B-27、ヘパリンナトリウムを補充した。必要に応じて可能な汚染のリスクを最小限にするために100 U / mlのペニシリンおよび100μg/ mlのストレプトマイシンの濃度で培地に抗生物質を加える。 </li><li>ソートRFP +上から調製した96ウェルプレートにRFP-細胞を、ウェルあたり1細胞（単球、細胞ベースアッセイ）及びウェル当たり100細胞（多細胞ベースの球アッセイ）。 100μノズル、シース圧力20 PSI、および歩留まりマスク0、純度マスク32、位相マスク16：シングルセルモード、ソートセットアップを使用して（ 材料を参照）ソートで市販のセルソーターを実行します。 </li><li>多細胞ベースのアッセイのために（顕微鏡的（単一の細胞ベースのアッセイのために）細胞を含むウェルをスコアによって個々のウェル中の細胞数を計数することにより、めっき効率を評価する; 図2 </stronG&gt;）。 </li><li> 37℃、5％CO 2で（組成についてステップ2.6参照）培地球における標準条件下で細胞をインキュベートする。毎日のbFGF（20ng / ml）およびEGF（20ng / mlの）を補完する。 </li><li> 1週間後、統合されたレポーターシステムからの蛍光シグナルを検出するための4倍または10倍の倍率蛍光顕微鏡で標準的な顕微鏡を使用して、新たな腫瘍球の数を数える。 「大」球としては100μmを超える直径の球をカウントし、直径50球- &quot;小さな&quot;球（ 図3）としては100μm。あなたが本当の球をカウントし、クラスターをセルではないことを確認してください。 注：単一のセルベースアッセイでは、球形成は文化の10（7対）日後に顕微鏡的に得点する方が簡単です。 </li><li> RFPの+中球形成細胞の割合を計算し、それぞれの単一細胞ベースのアッセイ（各個々の実験のために1枚の96ウェルプレート）または多細胞学士でRFP-細胞ショーらによって提示されたsed球アッセイ（各個々の実験用のウェル）。16 注：細胞を形成する球の割合（％）=（球の数）/（播種した細胞数）×100 </li></ol>球の3連続継代<ol><li> RTで10分間300×gで、適切な滅菌チューブと遠心分離機における各ウェルのコンテンツを配置します。マルチセルベース球アッセイのために、1つのウェルから球を一緒に集める。 PBSおよび2分より長い遠心分離機で5回 - よく3を洗う。単一細胞ベースの球アッセイのために、個々の球を集める。起因する細胞の数が少ないために、遠心分離および洗浄工程では1.5 mlチューブを使用しています。 </li><li> 0.05％トリプシン-EDTAの200μlの上清と再懸濁ペレットを削除します。 </li><li>最適な細胞分離を達成するために、ソフトシェーカーで5分間、37℃で細胞懸濁液をインキュベートする。細胞死率を下げるためにあなたの細胞株について、トリプシン処理時間を最適化：なし大型た場合球は穏やかに100μlのピペットチップを用いて可視粉砕物である。ケースで大球は、まだ存在している別の3分のトリプシンでインキュベートした後、トリチュステップに進みます。シングルセルベースアッセイの場合、トリプシン処理工程中の生きた細胞の最適な細胞収量のための時間を最適化するようにしてください。 </li><li>トリプシンを不活性化するために、単一細胞アッセイの場合には、10分間、500μlのに追加の2分間の遠心分離を300×gで遠心分離し、完全培地を加える。 </li><li>球培地に注意深く上清および再懸濁細胞を除去する。 </li><li>単一細胞懸濁液を得るために、40μmのセルストレーナーキャップフィルタを使用する。 </li><li> RFP +および多細胞bassedアッセイにおいてRFP-細胞を用いる場合には、フローサイトメーター分析を介して十分に再懸濁した後、各蛍光細胞の割合を評価する。 </li><li>上記で詳述したように調製した単一細胞の連続再プレーティングアッセイ、シードウェルあたり1細胞の新しい超低付着の96ウェルプレートに用。 1個々の球体からは、約20個々のウェルをシード。マルチセルベースの​​プライマリ球の再プレーティングアッセイのために、種子細胞を新しい96ウェルプレートに一次球の1つのウェルから得られ、顕微鏡法によって細胞数を翌日カウント。 </li><li> ステップ2.11で説明した式を用いた二球アッセイにおける球形成細胞の割合を評価する。 </li></ol> 4.結果解析<ol><li>独立した三連で行われた実験の結果を分析し、統計分析のために、正規分布の値とそれ以外のマン - ホイットニー·テストを分析するために、両側スチューデントのt検定を使用しています。 </li></ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cancer+stem+cell-like+cells+from+human+epithelial+ovarian+carcinoma+cell+line.">Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line.</a> Cell Mol Life Sci. 67 (22), 3915-3925 (2010).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+ovarian+cancer-initiating+cells+from+primary+human+tumors.">Identification and characterization of ovarian cancer-initiating cells from primary human tumors.</a> Cancer Res. 68 (11), 4311-4320 (2008).</li><li>Liang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxic+microenvironment+upgrades+stem-like+properties+of+ovarian+cancer+cells.">The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells.</a> BMC Cancer. 12, 201 (2012).</li></ol>

The authors have nothing to disclose.

球培養物は、癌幹細胞は、潜在的なアッセイおよびヒト腫瘍細胞15,25,26広範囲の幹様細胞を濃縮するために広く用いられている方法である。これらの培養条件下で、自己複製能を欠く癌細胞は分化し、最終的に細胞死を受けることが予想される。彼らは当初、特に第一のアッセイであっても腫瘍球を細胞クラスターを形成するか、またはかもしれないが、それらは、自己複製の特性の...

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 height="21" style="height:21px;">low-Attachment-plate&#160;</td>
			<td>Corning</td>
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			<td height="21" style="height:21px;">MEGM</td>
			<td>Lonza</td>
			<td>CC-3151</td>
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			<td height="21" style="height:21px;">Insulin</td>
			<td>Lonza</td>
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			<td>Lonza</td>
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			<td>SingleQuots&#8482; Kit</td>
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			<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 height="21" style="height:21px;">FGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>end concentration: 20 ng/ml</td>
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			<td height="21" style="height:21px;">B-27</td>
			<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 height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td style="width:119px;">15140-122</td>
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			<td height="21" style="height:21px;">FCS&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">10500-064</td>
			<td></td>
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			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Gibco</td>
			<td style="width:119px;">21875-034</td>
			<td></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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		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s PBS (1x)</td>
			<td>Gibco</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<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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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td>Gibco</td>
			<td style="width:119px;">21041-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12 (powder)</td>
			<td>Gibco</td>
			<td style="width:119px;">42400-010</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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.

従来の球アッセイにおいて、RFP + OVCAR-3細胞の約40％対RFP-細胞の20％が一次球アッセイにおける個々の腫瘍球（ 図4A）を生じた。また、RFP +細胞によって形成球体はRFP-細胞によって形成されたものよりもサイズが大きかった。 単一細胞に基づくアッセイでプレーティングすると、RFP +細胞はまた、上記の結果を確認し、RFP-細胞より球を形成した。しかし、多細...

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.

マルチとシングルセルベース球アッセイを用いてヒト卵巣癌細胞における幹細胞特性の評価

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 ビュー.  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.