この作品は脳腫瘍研究のためのフロリダセンター、脳腫瘍治療と米国立衛生研究所（NIH）のためにプレストンA.ウェルズ·ジュニア·センター（1R21CA141020-01）によってサポートされていました。

1。膠芽腫単一の細胞懸濁液を準備する<ol><li> Gliomasphere文化は前述の2,11,12のようにニューロスフィアアッセイ（NSA）を使用して確立され、維持されます。 </li><li> gliomaspheresを継代するための適切な時期に、球を含む培地を除去し、適切なサイズの滅菌組織培養チューブに入れ、室温で5分間800回転（110 g）で遠心分離した。 </li><li>上清を廃棄し、球のペレットを、0.05％トリプシン-EDTA 1mlに再懸濁し、37℃で3〜5分間水浴中でインキュベートする。 </li><li>大豆トリプシンインヒビターの等量はその後トリプシン活性を停止するために使用されています。 </li><li>細胞懸濁液を穏やかにトリプシン活性の均質性と完全な中和を確実にするために上下にピペッティングします。 </li><li>細胞懸濁液を再び5分間800rpmで遠心分離する。上清を除去し、細胞をNeuroCultの1ミリリットル中に再懸濁されるNSCの基礎培地。 </li><li>単細胞懸濁液の10μlのは、細胞数を数えるために、トリパンブルー90μlと混合される。 </li></ol> 2。携帯トレースカルボキシスクシンイミジルエステル（CFSE）緑色蛍光色素で染色<ol><li>染色されたように各1万個の細胞では、染色試薬1mlを5μMの最終染色濃度を与えるためにNeuroCult NSC基礎培地1mlに5 MM細胞トレースCFSE色素（Molecular Probes社、Invitrogen社製）1μlを添加することにより調製される。 </li><li>細胞を含む染色試薬が均一になるまで十分に混合し、10分間室温でインキュベートする。 </li><li>インキュベーションの間、氷のように冷たいNeuroCult NSCの基礎培地は、急冷溶液として調製されています。 </li><li>染色プロセスを停止するには、氷のように冷たい焼入れ液の約5-10ボリュームがロードされたのCFSE細胞に添加される。 </li><li>次いで、この溶液を5分間800rpmで遠心分離し、上清を廃棄する。 </li><li>目でポイントは、染色が正常に行われたことを示す、色の鮮やかな緑の色合いでチューブの底に細胞のペレットが表示されるはずです。 </li><li>細胞は、5分と上清を廃棄し800rpmで遠心分離、新鮮NeuroCult NSC基礎培地中1-2 mlにペレットを再懸濁することにより洗浄する。この手順を3回洗浄し、合計で繰り返されます。 </li><li>第三洗浄後、細胞をNeuroCult NSC基礎培地と細胞数行われ1mlに再懸濁する。 </li><li> CFSE染色された細胞は、その後、[50,000細胞/ ml]を適切な密度で滅菌組織培養フラスコに播種し、37℃/ 5％CO2の加湿インキュベーター内に配置されます。 </li></ol> 3。 CFSEローディング検証<ol><li> NSAの文化に播種した細胞をCFSE染色を検証するための蛍光顕微鏡下で監視することができます。この段階では、すべてのセルは明るい緑色（ 図1）を示す。から細胞懸濁液の代わりに、ドロップCFSE染色した細胞を顕微鏡スライド上に配置し、蛍光顕微鏡下で細胞を可視化するためにカバーガラスで覆われている可能性があります。 </li><li>成功したCFSE標識はさらに、フローサイトメトリーを介して確認された。各グループ1から5×10 5個の細胞のように多くはCFSE染色の検証のために十分である。 CFSEロードされた細胞と無負荷陰性対照細胞を500μlの合計ボリュームに対して、1つのx-PBSまたはNeuroCult NSC基礎培地中に再懸濁させ、それぞれ別々のFACSチューブ内に配置されます。 </li><li>フローサイトメーターは、関連パラメータのためにそのユーザーマニュアルの指示に基づいて調整される。 CFSEは492 nmと517 nmの発光極大波長の励起極大を有する。 </li><li>まず、アンロードされたコントロール細胞が実行され、取得されたイベントは、前方および側方散乱光（FSC対SSC）とヒストグラムプロットに記録され、主細胞集団（ 図2A）ゲートされます。 </li><li>同様に、CFSEロードされた細胞は、同じパラメータを使用して分析されるsコントロール細胞（ 図2B）に使用される。 </li></ol> 4。遅い分割の分離蛍光活性化細胞選別（FACS）を用いた細胞集団[擁すなわちCFSE]を<ol><li>分裂時には、CFSE強度が減少し、hGBM細胞が緑色蛍光強度（ 図3）のさまざまなレベルを示す細胞から構成されているgliomaspheresを形成しています。 gliomaspheresの直径は約150から200ミクロン（約5〜10日で細胞株の成長率に応じてポストCFSEローディング）の平均サイズに達したら、球を含む培地を除去し、適切なサイズの滅菌組織培養に配置され管し、室温で5分間800rpmで遠心分離した。 </li><li>前の手順で説明されて、細胞がアップミリリットル当たり10 7個の細胞の密度で細胞懸濁液を準備するために、細胞数を、PBS中に再懸濁されるような単一細胞懸濁液を調製する。 </li><li>アフトerはPBSの適切な音量で細胞を再懸濁し、ヨウ化プロピジウム（PI、Sigma社、500μg/ mlの）は、フローサイトメトリーによってソート/分析した場合、死細胞を除外するために細胞懸濁液の1μl/ mlの濃度で添加される。 </li><li>完全NeuroCult NSC媒体の各1ミリリットルを含む2つの15 mlの滅菌組織培養管はソートが遅い分裂細胞（CFSE保持する細胞）と高速分裂細胞（CFSE希釈細胞）を収集するために使用されます。 </li><li>まず、アンロードされた細胞群からの細胞懸濁液を、フローサイトメーターを介して実行されます。 </li><li>細胞（イベント）は、細胞の大部分が流れプロットに含まれるように調整されている前方散乱（FSC）の対側方散乱光（SSC）と両方のパラメーターの電圧パラメータに基づいてプロットされます。 </li><li>死亡または損傷した細胞を排除するために、細胞は、SSCに対し、PIの反応性に基づいてプロットされ、単一の生細胞集団は、PI（負）ゲートされています。 </li><li>その後、均一な細胞集団では、fに基づいて選択されorward散乱（FSC）の対側方散乱光（SSC）。 </li><li>均質な単一生細胞集団を対数目盛で設定されたセル周波数対CFSE強度に基づいてヒストグラムにプロットされます。 CFSEレーザー強度は、0〜100の間には負の信号を配置するために調整されます。 </li><li>同じパラメータと同様のゲーティング戦略を用いて、CFSE標識された細胞は、フローサイトメーターを介して実行されており（ 図4C）を分析。 </li><li>その後、細胞を、全体的な人口の遅い割る人口は（速く分裂している細胞）をCFSE（それぞれCFSE 高上位5％とCFSE 低ボトム85％）を保持する能力に基づいて識別されます。 </li><li>これらの異なる細胞集団は、その後、完全NSC媒体の1ミリリットルを含む個別の15ミリリットル滅菌組織培養管に分類されます。 </li><li>選別された細胞は、その後、5分間800rpmで遠心分離する。 </li><li>上清を捨て、ペレットを適切に再懸濁するpriate媒体の容量（単離されたイベントまたはペレットの大きさの数に応じて）とセル数が実行されます。 </li><li>異なるソート集団由来の細胞は、その後、適切な滅菌組織培養フラスコ内のミリリットル当たり50,000個の細胞を播種し、連続的に継代または下流の実験のために使用される前に5-10日を37℃/ 5％CO2の加湿インキュベーター内に配置されます。 </li></ol> 5。代表的な結果 CFSEをロードした後、すべての細胞が顕微鏡（ 図1）に基づく可視化などの強い緑色蛍光を示す。この緑がかった色があっても肉眼（ビデオ参照）で見ることができます。フローサイトメトリーでこれらの細胞を分析することも、コントロール細胞（ 図2）と比較して非常に強烈な緑色の蛍光を示す。ニューロスフェア培養におけるめっきCFSE細胞読み込まれると、これらの細胞は増殖し、5〜10日間で150から200μmのgliomaspheresを形成している。細胞は、トンを増殖として<html>彼蛍光染料が均等にすべての細胞分裂による蛍光強度の半減につながる、部門により娘細胞の間で分配される。膠芽腫は、機能的に異質であり、異なる時間スケールで増殖している細胞で構成されています。このプロトコールに記載された方法は、それぞれのCFSEを希釈または保持する能力（ 図3、また、ビデオを参照）のおかげで、高速と低速の分裂細胞を同定および単離するために1つを可能にします。 アンロードされた細胞と比較してのCFSEロードされた細胞から生成解離gliomaspheresのフローサイトメトリー分析は、多様なCFSE保持特性（ 図4）を示しています。ファスト分裂細胞（下85％）または低速分裂細胞（上位5％ - 高 CFSE）ニューロスフィアアッセイ条件（ 図5）で培養したときの能力を形成する展示gliomapshere。 グレ1 &quot;/&gt; 図1解離腫瘍細胞直ちにCFSE色素をロードした後。細胞が正常なラベリングを示す鮮やかな緑色を呈する。元の倍率、10×。 <img src="/files/ftp_upload/3918/3918fig2.jpg" alt="図2" /> 図2：フローサイトメトリーを用いてCFSEローディング確認。）無負荷細胞、B）CFSEは、ロードされた対無負荷の細胞でCFSE蛍光強度を比較すると）細胞とCをロードしました 。標識および非標識細胞との間の蛍光強度の大きさの違いのいくつかの注文があることに注意してください。 <img src="/files/ftp_upload/3918/3918fig3.jpg" alt="図3" /> 図3：6日間めっき後のCFSEロードされた細胞に由来する代表gliomasphere。いくつかの細胞は非常に明るく、CFSE染色を示す。元の倍率、63×。 <imグラムSRC = &quot;/ files/ftp_upload/3918/3918fig4.jpgの&quot; alt = &quot;図4&quot; /&gt; 図4代表的フローサイトメトリープロット/ 6日CFSE染色後に高速と低速に分裂している細胞を単離するためのヒストグラム。死亡または損傷を受けた細胞を除外するための全体の細胞集団を表示する）FSC対SSC、B）は 、SSC対PIの反応性、 C）のゲートにFSC対SSC均質生きた細胞集団、D）は 6日、陰性対照と比較して大きい蛍光強度によって証明されるようにCFSE標識した後、CFSE保持する細胞が存在することを示します。ヒストグラムもCFSE強度が時間とともに減衰していることを示しています。E）は CFSE保持する細胞（上位5％）およびCFSE希釈セル（ボトム85％）を識別するためのゲーティング戦略を表示するヒストグラム。 <img src="/files/ftp_upload/3918/3918fig5.jpg" alt="図5" /> 図5から発生する代表gliomaspheres <stronG&gt; A）の高速分裂細胞（CFSE 低 ）とB）の遅い分裂細胞（CFSE 高 ）。元の倍率：それぞれ10倍と20倍C）が代表細胞数が通過時に速いか遅いか分裂細胞由来の培養物から得られる。 P &lt;0.05、t検定。 <a href="http://www.jove.com/files/ftp_upload/3918/3918fig5large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

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研究数の増加は、腫瘍形成、治療抵抗2月8日に貢献癌の細胞の遅い分割するサブコンパートメントの重要性を実証している。したがって、それは別の成長率と集団を比較するために、より一般的に細胞またはこの特定の亜集団の研究を可能にする実験方法を記述し、標準化することが重要です。細胞分裂の彼らの率に基づいて、細胞の亜分画を同定および単離するために、CFSEを使用?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> NeuroCult NSC基礎培地（ヒト） </td><td>培地</td><td>電池技術を食い止める</td><td> 05750 </td></tr><tr><td> NeuroCult NSCの増殖サプリメント（人間） </td><td>培地サプリメント</td><td>電池技術を食い止める</td><td> 05753 </td></tr><tr><td> 0.05％トリプシン-EDTA </td><td>試薬</td><td>ギブコ</td><td> 25300-062 </td></tr><tr><td>ダイズトリプシン阻害因子</td><td>試薬</td><td>シグマ</td><td> T6522 </td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>試薬</td><td>ギブコ</td><td> 15140-122 </td></tr><tr><td> T25フラスコ</td><td>文化ウェア</td><td> Nalgeヌンク国際</td><td> 136196 </td></tr><tr><td> T80フラスコ</td><td>文化ウェア</td><td> Nalgeヌンク国際</td><td> 178905 </td></tr><tr><td> 15 mlチューブ</td><td>文化ウェア</td><td> BDファルコン</td><td> 352096 </td></tr><tr><td> 50mlチューブ</td><td>文化ウェア</td><td> BDファルコン</td><td> 352070 </td></tr><tr><td> EGF </td><td>増殖因子</td><td>研究開発</td><td> 2028-EG </td></tr><tr><td> CellTrace CFSE細胞増殖キット</td><td>蛍光細胞分裂が追跡用色素</td><td> Molecular Probes社、Invitrogen社</td><td> C34554 </td></tr></tbody></table>

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

腫瘍の不均一性は、腫瘍の堅牢性を支える基本的な特徴を表しており、治療戦略1の発展に中心的な障害となる。腫瘍の不均一性の問題を克服するためには、特定の疾患の病態を推進し、臨床的に関連するターゲットを表す腫瘍集団の表現型、（エピ）の遺伝的および機能的同定および特徴付けを可能にするアッセイおよびツールを開発することが不可欠である。それは、腫瘍は細胞分裂の周波数の異なる細胞の異なる亜分画を示すこと、および低速サイクリングという機能的な基準を積極的に脳を含めたいくつかの癌における腫瘍形成能力に関連付けられていることを今では十分に確立されている乳房、皮膚、膵臓ならびに白血病2-8。蛍光色素カルボキシスクシンイミジルエステル（CFSE）は、in vitroおよび血液媒介トンを用いたin vivo での細胞の分裂頻度を追跡するために使用されてきたumorsや膠芽腫などの固形腫瘍2,7,8。 CFSEの細胞透過性の非蛍光プロドラッグは、安定したアミド結合9を形成する細胞内のアミン基とのスクシンイミジル部分との反応によってタンパク質と共有結合することにより細胞内に保持される蛍光化合物に細胞内エステラーゼによって変換されます。蛍光染料が均等にすべての細胞分裂による蛍光強度の半減につながる、部門により娘細胞の間で分配される。これは、部門10の8〜10ラウンドまでの細胞周期の周波数のトラッキングが可能です。 CFSE保持能力は、癌幹細胞活性2に濃縮されることが実証遅いサイクリング亜集団（上位5％の色素保持する細胞）を同定し、単離する脳腫瘍細胞で使用されました。 このプロトコルは、人間の文化の中でglio CFSEで細胞を染色する技術と、個々の集団の単離を記載芽腫（GBM）の由来のフローサイトメトリー2を使用して異なる分裂速度 ​​を有する細胞。技術はCFSE標識を保持する能力によって細胞の脳腫瘍スローサイクリング人口を同定および単離するのに役立っています。

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

このビデオプロトコルは、細胞分裂の頻度に基づいて、ヒト神経膠芽腫の細胞の異なるサブ集団の同定と分離するための蛍光色素カルボキシスクシンイミジルエステル（CFSE）のアプリケーションを示しています。

カルボキシフルオレセインスクシンイミジルエステル（CFSE）を用いてヒト神経膠芽腫のスロー非分裂細胞の同定および単離

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

identification-isolation-slow-dividing-cells-human-glioblastoma-using

Research

JoVE Journal

Medicine

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

14.0K ビュー.  The University of Florida. このビデオプロトコルは、細胞分裂の頻度に基づいて、ヒト神経膠芽腫の細胞の異なるサブ集団の同定と分離するための蛍光色素カルボキシスクシンイミジルエステル（CFSE）のアプリケーションを示しています。

ビデオ: 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.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

医学

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.

ヒト前立腺癌サンプルからがん幹細胞の単離

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.

臨床応用のためのヒト羊膜上皮細胞の単離、凍結保存と文化

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.

マルチパラメータ蛍光活性化細胞選別によるヒトリンパ内皮細胞の単離

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.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

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.

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

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.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

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.

マウスモデルにおける膠芽細胞腫に対する治療用幹細胞の鼻腔内送達

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.

フローサイトメトリーを用いたひと脂肪組織での免疫細胞のキャラクタリゼーション

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.

用語胎盤の胎児膜から主のひと脱落膜細胞の分離

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