This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

以下で説明するプロトコルは、UIUC人間研究倫理委員会のガイドラインに従っています。 1.収集と外科組織サンプルの消化<ol><li>右結腸切除（ 図1Aおよび1B）後の腫瘍組織サンプルを採取します。同様に、隣接する健康な部位から組織を採取してください。 </li><li> 12ミリリットルのHBSS溶液を含有する15ミリリットルバイアルにすぐに組織を転送します。絶縁発泡ボックス内の氷の上にバイアルをしてください。 </li><li> 45分以内に、さらなる処理のために、組織培養フードにバイアルを含む組織を輸送します。フード内部の氷のブロックにバイアルをしてください。 </li><li>ピペットを用いて、HBSS溶液を6-7 mlを含む6ウェルプレート内に供給された組織を注ぎます。 注：ウェル当たり組織の量は、このすすぎ工程で重要ではありません。 </li><li>氷のブロックに6ウェルプレートにしてください。二回HBSS溶液中で組織をすすぎます。 </li><li>無菌SCISと分離セクションにきれいに組織を切断SOR。小さ ​​なセクションにミンチ組織滅菌メスの刃でサイズ1〜2 mm 3でより大きくありません。 </li><li>組織を転送する前に（0.1％トリプシン溶液1 mlを含む）標識された0.1％のトリプシンバイアルを秤量します。ピペットで0.1％のトリプシンバイアルのそれぞれに〜20mgの組織を転送します。 </li><li>トリプシンのさらなる希釈を回避するために、トリプシンバイアルにHBSSの移動を最小限に抑えるようにしてください。 </li><li>組織転送後、トリプシンバイアルを計量。組織塊のような違いを文書化します。 </li><li> 24ウェルプレートの各ウェルにトリプシン〜20mgの組織と1ミリリットルを注ぎます。 </li><li> 16〜20時間4℃の冷蔵庫でシェーカー上で24ウェルプレートを置きます。この待機期間中又は早期に、ポリアクリルアミドゲル基材を準備し、セクション3で説明したようにECMタンパク質とそれらを官能。 </li></ol>単一細胞懸濁液に2組織試料の酵素的解離およびECM官能弾性Substratの単離された細胞を培養しますESと硬質ポリスチレン料理<ol><li>シェーカー上で4℃でよくトリプシンでのインキュベーションの16〜20時間後、ピペットを用いて、（完全な増殖培地2-3 mlを含む）4°C完全な増殖培地バイアルに4℃のトリプシンから組織を移します。増殖培地へのトリプシンの最小限の移動を確認してください。 注：完全増殖培地処方は以下の通りである：10％ペニシリン - ストレプトマイシン溶液全体の1％の最終濃度までウマ血清を補充したRPMI 1640基本培地。 </li><li> 12〜15分間37℃の温水浴中でメディアバイアルを含む組織を配置します。 </li><li>ピペットを用いて、HBSS溶液を含有する15ミリリットルバイアルに組織を移し、静かに振ります。 </li><li> 2.3を繰り返します。 </li><li> HBSS溶液を、1mlの溶液を含むバイアル（HBSS）で転送〜0.1％コラゲナーゼ溶液から組織を削除します。加湿細胞培養インキュベーター中で37℃、5％CO 2環境で45分間インキュベートします。待機期間、暖かいgrowt中に15ml中のH培地を水浴中で37℃にてバイアル。 </li><li>コラゲナーゼのキャリーオーバーを最小限にピペット内にすべての組織片を引き出します。予熱した培地バイアルにちょうど組織片を取り出します。 </li><li> 6ウェルプレートの各ウェルに40ミクロ​​ンよく挿入フィルタを配置します。 1mLの細胞培養培地を用いてフィルターを湿ら。 </li><li>組織が完全に解離されるまで、10mlのガラスピペットで数回メディアでトリチュレート組織。 </li><li>バイアルの底にできるだけ近いピペットチップを維持します。 </li><li>破片や未解離部分を除去するフィルター上に溶液を堆積させます。 </li><li>遠心分離は、室温で5分間、150×gで培地中に細胞懸濁液を濾過しました。 </li><li>その後上清を除去し、2mlの新鮮な培地で細胞ペレットを再懸濁します。 </li><li> （必要な場合）血球計数器を用いて細胞数を計測します。シードは、細胞外マトリックス（ECM）官能ゲルおよび所望の濃度のポリスチレンディッシュ上で初代細胞を単離しました。</li></ol>異なる剛性ポリアクリルアミド（PA）ゲルおよびポリスチレン基質の調製と機能化注：第3の視覚的なデモでは、視聴者/読者は、最近の可視化実験のジャーナル（Joveの）第17条に言及されています。 <ol><li>採用公開プロトコル18、19は 、12ミリメートル2ガラスカバーは、ハイドロゲルの共有結合を確実にするために、化学的にスリップ有効にしてください。 <ol><li>まず、ガラスカバーは、室温で7分間、3- Aminopropyltrymethoxysilane（ATS）でスリップ扱います。 </li><li> DI水リンスで完全にATSを削除して、治療のカバーは（70％グルタルアルデヒドのストック溶液からPBSで希釈）の0.5％グルタルアルデヒドで30分間溶液を用いてスリップ。 </li></ol></li><li> / vのアクリルアミド溶液および0.05％N、N &#39;-methylenebisacrylamide（ビス）10mMのHEPES緩衝化生理食塩水20内の溶液wの5％を混合して2キロパスカルのゲル溶液を得ます。 Vアクリルアミドおよび0.13 / wの8％を使用してください10 kPaのゲル20のための10mMのHEPES緩衝生理食塩水で％N、N &#39;-methylenebisacrylamide（ビス）ソリューション。 <ol><li> 200過硫酸アンモニウム（10％w / v）を1：両方の場合において、1を使用して2,000 N、N、N &#39;、N&#39;はそれぞれ、重合プロセスの開始剤および触媒として-tetramethylethylenediamine（TEMED）。 </li><li>また、受託者のマーカーとして2 kPaのゲル溶液に21、22を 100μlの蛍光ビーズを追加します。 </li></ol></li><li> 12ミリメートル2活性化ガラスカバースリップ上に20μlのプレポリマーPAゲル溶液の液滴を付着させます。ドロップで別の12ミリメートル2正規ガラスカバースリップを置きます。ドロップが毛細管現象によるカバーガラスの間に広がっていることを確認してください。ビーズの大部分は細胞培養表面の近くに来ることを確実にするために2 kPaのゲルのためのサンドイッチを反転。 </li><li> RTで45分間、PAゲルを治します。 </li><li>単一のエッジかみそりを使用して、トップカバースリップをはがし。 注：一方の端から剥離、剥離が進行中にサンドイッチの。ゲルは、活性化されたスライドガラスに付着したままです。 </li><li>使用前にPBS溶液中にゲルを保管してください。 </li><li> ECM分子は、公開された方法23以下の50μg/ mlの濃度でヒトフィブロネクチンとPAゲルおよびガラスを官能。 <ol><li>簡単に言えば、2ミリリットルの純粋なヒドラジン水和物のO / Nを有する基板をインキュベートします。 </li><li>ヒドラジン水和物を除去し、脱イオン水で十分に基板をすすぎます。 </li><li> 30分間、5％酢酸で基板を洗浄します。 </li><li>酢酸を除去し、脱イオン水で十分に基板をすすぎます。 </li><li> 30分間DI水に浸漬した基板を保管してください。 </li><li> 50μg/ mlの濃度で35分間酸化フィブロネクチンを有する基板をインキュベートします。 </li><li> 10分間の低回転でシェーカー上でPBSで基板を洗浄します。 </li><li>細胞をプレーティングする前に30分間、2〜3 mLの培養培地中で、37℃で、すべての基質をインキュベートします。 注意：プレートの細胞をまばらに移入にD方式（1,000〜3,000細胞/ cm 2）。各ゲルで覆わ​​れたガラススリップを35mmペトリ皿中に含有させる必要があります。細胞は、任意の顕微鏡（少なくともO / N）の前に完全に接着させます。 </li></ol></li></ol> 4.牽引力顕微鏡および免疫蛍光顕微鏡アッセイ<ol><li>牽引力顕微鏡アッセイ<ol><li>ウォーム0.25％トリプシンEDTA / 10分間水浴中で37℃で10％SDS溶液を牽引実験を開始する前に。 </li><li>蛍光倒立顕微鏡ステージ（32X倍率）で、時間と場所で細胞培養インキュベーターから1つのゲルを削除してください。 </li><li>視野内の1つのセルを検索します。ペトリ皿のふたを外します。 </li><li>細胞の位相コントラスト画像（ 例えば 、 図3）を取ります。 </li><li>蛍光に撮影モードを切り替えて、適切なフィルタを選択します。この時間の間に、顕微鏡ステージまたはサンプルを動かさないでください。 </li><li>蛍光ビーズの画像を取るdisplac携帯牽引（ 図4C）によって編。 </li><li>ゲルからの細胞を分離するためにペトリ皿に1ミリリットルのトリプシン/ SDS溶液を追加します。この時間の間に、顕微鏡ステージまたはサンプルを動かさないでください。また、ゲルからの完全な除去を確実にするために、セルの制御画像を取ります。 </li><li>細胞を除去した後、ビーズの基準（ヌル力）の画像を取ります。 </li><li>他のすべてのゲルのための手順を繰り返し4.1.2-4.1.8。 </li><li>各ケースのステップ4.1.6と4.1.8で取得した二つの画像からのImageJを用いた画像スタックを作成します。スタックするスタック→画像→画像：画像スタックを生成するには、画像を開いた後のImageJで次の一連のコマンドを使用します。 </li><li> 。変位場とトラクションを得るためには、公開された方法21、22次のImageJのプラグインを使用して次のリンク使用して、これらのプラグインのコードと詳細なチュートリアルを入手： <a href="https://sites.google.com/site/qingzongtseng/imagejplugins">https://sites.google.com/site/qingzongtseng/imaを</a>gejplugins。 <ol><li> 「テンプレートマッチング」プラグインを使用して、スタック内の画像の位置を合わせます。 OKプラグインスタックに→整列スライスマッチング→テンプレート→：ステップ4.1.10で生成された画像スタックを開いた後のImageJで次の一連のコマンドを使用します。その結果、画像スタックを保存します。次の手順で、この新しい画像スタックを使用します。 </li><li> PIV（粒子画像流速）プラグイン（ 図4D）を使用して変位場を取得します。 [OK]を→→OK→このPIVおよび出力を受け入れプラグイン→PIV→反復PIV（基本）：ステップ4.1.11.1に保存された画像スタックを開いた後、次の一連のコマンドを使用します。元の画像スタックと同じディレクトリにPIV出力を保存します。次のステップで入力としてこれを使用してください。 </li><li>最後に、FTTC牽引マップ（ 図4E）を得るためにプラグインを（フーリエサイトメトリートラクションを変換）を使用します。プラグイン→FTTC→イン：次の一連のコマンドを使用しますERTの材料特性、 すなわち 、ヤング率およびPAゲルのポアソン比→OK→OKステップ4.1.11.2→で保存したPIV出力ファイルを選択します。保存FTTCは、画像スタックとPIV出力と同じディレクトリになります。 </li></ol></li></ol></li><li>免疫蛍光顕微鏡アッセイ<ol><li>層流フードに免疫染色する基材を取り出し、培養培地を除去します。 </li><li> PBSで基板を洗浄し、室温で20分間、PBS中の4％パラホルムアルデヒドで細胞を固定します。 </li><li> 3回（各5分の時間）のために、PBSで基板を洗浄します。その結果、30分間500μlのシグナルエンハンサーを有する基板をインキュベートし、PBSですすいでください。 </li><li> RTで45分間、PBS中で250倍に希釈1のモノクローナル抗ビンキュリン抗体で細胞をインキュベートします。 3回（各5分の時間）のために、PBSで基板を洗浄します。 </li><li> 200希釈：1で二次抗体Alexa Fluor 488ヤギ抗マウスIgGとサンプルをインキュベート室温で30分間PBS中。 3回（各5分の時間）のために、PBSで基板を洗浄します。 </li><li> F-アクチンの構造を可視化するために、TRITCをRTで45分間濃度を50μg/ mlのコンジュゲートをファロイジンで細胞を培養します。 3回（各5分の時間）のために、PBSで基板を洗浄します。 </li><li>イメージ共焦点走査型レーザー顕微鏡を用いた試料（ 図5（a） - （d））。 </li></ol></li></ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+hydrogel+substrates+with+tunable+mechanical+properties.">Preparation of hydrogel substrates with tunable mechanical properties.</a> Current Protocols in Cell Biology. , 10-16 (2010).</li><li>Tseng, Q., 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=Spatial+organization+of+the+extracellular+matrix+regulates+cell&#8211;cell+junction+positioning.">Spatial organization of the extracellular matrix regulates cell&#8211;cell junction positioning.</a> Proc. Natl. Acad. Sci. USA. 109 (5), 1506-1511 (2012).</li><li>Chopra, A., 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=Augmentation+of+integrin-mediated+mechanotransduction+by+hyaluronic+acid.">Augmentation of integrin-mediated mechanotransduction by hyaluronic acid.</a> Biomaterials. 35 (1), 71-82 (2014).</li><li>Damljanovic, V., Lagerholm, B. C., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+and+micropatterned+conjugation+of+extracellular+matrix+proteins+to+characterized+polyacrylamide+substrates+for+cell+mechanotransduction+assays.">Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays.</a> Biotechniques. 39 (6), 847-851 (2005).</li><li>Tilghman, R. W., 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=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PLoS ONE. 5 (9), e12905 (2010).</li><li>Ulrich, T. A., de Juan Pardo, E. M., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+rigidity+of+the+extracellular+matrix+regulates+the+structure,+motility,+and+proliferation+of+glioma+cells.">The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells.</a> Cancer Res. 69, 4167-4174 (2009).</li><li>Williams, B. J., Anand, S. V., Rajagopalan, J., Saif, M. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-propelled+biohybrid+swimmer+at+low+Reynolds+number.">A self-propelled biohybrid swimmer at low Reynolds number.</a> Nat. Commun. 5, (2014).</li></ol>

The authors have nothing to disclose.

携帯牽引ストレスが最近転移状態13の潜在的な生物物理学的指標として浮上しています。しかし、原発腫瘍細胞との実験牽引データは、これまで文献に存在しません。また、直接異なる剛性のポリアクリルアミドゲル上で分離された原発性結腸細胞を培養することはまだ報告されていません。したがって、我々は、ゲル、ポリスチレン（ 図2）に最適化された原発性結腸?...

機械的な微小環境は、生物化学的要因に加えて、細胞機能の調節において重要な役割を果たしていることが、近年では、ますます明らかになっています。細胞を検知し、それらが3-7（2D培養におけるような）に付着または（3D培養においてなど）によって取り囲まれている基板の剛性に対応することができます。そうすることによって、細胞は、その分化3、形態4、遊走/運動性5、バイオ物性6、成長7、およびその他のプロセスを調節することができます。 癌細胞は、接着受容体と関連するシグナル伝達、膜タンパク質、細胞骨格アーキテクチャ、および分子モーター1,2の協調的、階層的なメカノケミカルの組み合わせを使用して2Dおよび3Dマトリックスの剛性に応答する。例えば、乳腺上皮細胞（のMEC）培養したときの剛性に似て150 Paの基板上に、通常の腺房実質を形成健康な乳房組織の。興味深いことに、彼らは腫瘍間質8の剛性を模倣するより硬い基板（&gt; 5,000 Pa）での培養の構造および転写の両方の開発腫瘍の顕著な特徴は、示します。また、別の実験では、乳房腫瘍形成は、コラーゲン架橋およびECM 9を補強することによって達成されることを示しています。最近の実験は、ヒト結腸癌（HCT-8）を示す細胞は、生理学的に関連する剛性（20から47キロパスカル）を有する2次元基質上で培養したときの表現型（MLP）のような転移を表示ではなく、上の非常に硬い（3.6万気圧）基板10- 12。これらの細胞最初のフォーム腫瘍様細胞クラスターとは、その後、周囲から出発し、互いから解離します。丸みを帯びた形態学（E R遷移）に変更が発生する上皮このように、それらは、細胞 - 細胞および細胞-ECM付着を低減し、増殖、および遊走になります。これらを示さない非常に難しいポリスチレン基板上のHCT-8細胞培養悪性形質。したがって、それがHCT-8細胞が原因で、適切な微小環境への暴露に転移性になると仮定されています。これらの実験は、初代ヒト癌細胞を、一次細胞由来不死化ガン細胞株またはマウスで行われていないことは注目に値します。 最近の研究では、細胞の増強牽引応力が転移細胞選択図13の研究のための潜在的な生物物理学的な署名として使用することができることを提案しているポリアクリルアミドゲル上の異なるヒト癌細胞株のための牽引力を測定することを含みます。これは、転移性癌細胞は、全てのケース13内の非転移性細胞と比較して有意に高い引張り応力を発揮することができることがわかりました。しかし、これらの結果は、直接に、マウス由来の乳癌細胞株14で先に公開された知見と矛盾します。また、最近の研究では、それらの細胞骨格のリモデリングのプロ中の不死化および初代ヒト細胞間の顕著な違いを強調しテインプロファイリングおよび細胞生存タンパク質発現15。したがって、主要なヒト癌細胞に対するトラクションを含む、生物物理学的アッセイの多くを再検討することが重要です。これは、一次細胞が不死化癌細胞株の牽引傾向を再現するかどうかの質問に対処します。 ここで説明するプロトコルは、ソフト基板（ポリアクリルアミドハイドロゲル）などのペトリ皿の上にそれらを培養する（健常および癌両方）初代ヒト結腸細胞の単離のために最適化されています。プロトコルは、消化し ​​、単一細胞懸濁液16に外科的に組織サンプルの結果としての酵素的解離に基づいています。我々の知る限り、これは直接サイトメトリー牽引用の組み込み蛍光マイクロビーズとソフトヒドロゲル基材上に分離された原発性結腸腫瘍および正常細胞を培養の最初の実証です。透明なゲル基質はまた、免疫染色を可能にします。このアッセイは、F-アクチン組織の違いを明らかにし基板剛性の変化に応じて一次ヒト結腸細胞における接着斑。この細胞培養プラットフォームは、癌の予後のためのパラメータとして、細胞剛性とトラクションのような一次ヒト細胞の様々な生物物理学的特性を探索の可能性を開きます。

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	<tbody>
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr >
			<td >HBSS</td>
			<td>Life technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td>Worthington</td>
			<td>LS003736</td>
			<td></td>
		</tr>
		<tr >
			<td >Collagenese</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr >
			<td >3- Aminopropyltrymethoxysilane (ATS)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td >281778</td>
			<td></td>
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		<tr >
			<td >Glutaraldehyde</td>
			<td>Polysciences, Inc.</td>
			<td>01201-5</td>
			<td></td>
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		<tr >
			<td >Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
			<td></td>
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			<td >N- methylenebisacrylamide (bis)</td>
			<td>Sigma-Aldrich</td>
			<td>M1533</td>
			<td></td>
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		<tr >
			<td >HEPES buffer solution</td>
			<td>Sigma-Aldrich</td>
			<td >83264</td>
			<td></td>
		</tr>
		<tr >
			<td >Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
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		<tr >
			<td >N&#714;-tetramethylethylenediamine (TEMED)</td>
			<td>Bio-Rad</td>
			<td>161-0801</td>
			<td></td>
		</tr>
		<tr >
			<td >Human fibronectin</td>
			<td>BD biosciences</td>
			<td >354008</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrazine hydrate</td>
			<td>Sigma-Aldrich</td>
			<td >18412</td>
			<td>Hazardous</td>
		</tr>
		<tr >
			<td >Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT15710</td>
			<td></td>
		</tr>
		<tr >
			<td >Signal enhancer</td>
			<td>Life technologies</td>
			<td>I36933</td>
			<td></td>
		</tr>
		<tr >
			<td >Monoclonal anti vinculin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V9131</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa fluor 488 goat anti-mouse IgG</td>
			<td>Life technologies</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr >
			<td >TRITC phalloidin conjugates&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr >
			<td >0.1 &#181;m fluroscent beads</td>
			<td>Life technologies</td>
			<td>F8801</td>
			<td></td>
		</tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Life technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr >
			<td >12 mm2 glass cover slips</td>
			<td>Corning</td>
			<td>2865-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

癌細胞は、接着受容体と関連するシグナル伝達、膜タンパク質、細胞骨格アーキテクチャ、および分子モーター1、2からなる協調階層メカノケミカル·システムを使用して複雑に機械的剛性をマトリックスに応答する。機械的感受性の異なる癌細胞のin vitroでされていますない主要なヒト癌細胞と不死化細胞株またはマウス由来の一次細胞で主に検討しました。したがって、ほとんどは、 インビトロで原発性ヒト結腸癌細胞の機械的感受性について知られています。ここで、最適化されたプロトコルは、健康及び癌の外科的ヒト組織サンプルからの初代ヒト結腸細胞の単離を記載が開発されています。単離された結腸細胞は、その後、（ソフト（2 kP​​aの剛性）と（10 kPaの剛性）、ポリアクリルアミドハイドロゲルと細胞外マトリックスによって官能硬質ポリスチレン（〜3.6 GPaの剛性）基板硬いフィブロネクチン上で正常に培養されますこの場合）。蛍光マイクロビーズは、細胞培養表面近く柔らかいゲルに埋め込まれ、かつ牽引アッセイはフリーオープンアクセスソフトウェアを使用して、細胞の収縮応力を評価するために行われます。また、異なる剛性基板上の免疫蛍光顕微鏡は、基板剛性の関数としての接着斑を含む一次細胞形態、細胞骨格の組織とビンキュリンに関する有用な情報を提供しています。

上述のプロトコルが正常に機関の審査委員会のガイドラインに従って四つの異なる患者からの複数の組織サンプル（N = 12）のために使用される。 図1Aは、右の細胞培養のための組織切片が得られるから、手術後の代表的な大腸の腫瘍を示しています。さらなる処理のために層状フードに移した後HBSS溶液中の代表的な組織切片を、 図1Bに示されています。単一細胞懸?...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

外科的組織からの初代ヒト結腸腫瘍細胞の単離およびトラクションサイトメトリーのためのソフト弾性基板上にそれらを直接培養

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Research

JoVE Journal

Bioengineering

15.4K ビュー.  University of Illinois at Urbana-Champaign. A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses. 

ビデオ: Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

患者の血液サンプルから生存循環腫瘍細胞の急速な分離

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

生物工学

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

電気刺激のマウス筋芽細胞前駆細胞およびアプリケーションからの工学骨格筋組織

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

ミササイトメトリー：CyTOFマスメーターで毎日のチューニングと実行した細胞サンプルのためのプロトコル

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

はSiO：フォトリソグラフィによって画定されたパリレン-C上の細胞のパターニング<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

外科用回収、単離と<em&gt;インビトロ</em&gt;組織工学アプリケーションのためのヒト前十字靭帯由来細胞の拡大

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

インデントと引張試験を用いたヒト軟部組織の生体力学的特性評価

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

フローサイトメトリーによる一次マウス網膜神経節細胞（RGCs）の単離

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人間歯槽骨膜から骨膜由来細胞の骨形成促進活性に及ぼすビタミン D の間葉系幹細胞の分離

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

広視野蛍光顕微鏡を用いたメカノバイオロジー研究の柔らかいシリコーン基板の剛性測定

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.