この仕事のための資金は、アメリカ心臓協会 (16SDG29870005) およびノースカロライナ州立大学研究とイノベーションのシード資金を提供されました。

1 です一日 1: セルと試薬調製 注: 生物学的安全キャビネットのサンプルの汚染を防ぐためにすべてのセルと試薬の準備を行わなければならない。。
<ol><li>暖かいフィルター ダルベッコ &#39; s 修正イーグル &#39; s 媒体 (DMEM)、ウシ胎児血清 (FBS)、L-グルタミン、37 ° C の水浴のペニシリン/ストレプトマイシン</li>。
	<li>10 %fbs、1% ペニシリン/ストレプトマイシン、1% と DMEM を温めて準備新生児ひと真皮線維芽細胞 (HDFn) メディアを補うことで L-グルタミン</li>。
	<li>は、凍結保存培地を取り除き冷凍の HDFns と 200 x g で 8 分間、遠心分離のバイアルを解凍します。T-75 培養用フラスコに 1 x 10 6 セル/mL と種子の密度で準備された HDFn メディアで細胞を再懸濁します</li>。
	<li>(37 ° C、5% CO 2) 細胞増殖を許可するインキュベーターで場所フラスコ</li>。
	<li>ストア HDFn のメディアで 4 ° C</li>
	<li>必要に応じてプロおよび反 migratory 試薬を準備します</li>。
</ol> 2。3 日目: 回転楕円体準備 <ol><li>サンプルの汚染を防ぐために文化フードで以下を実行します</li>。
	<li>電池が 80% の confluency に達するときメディアを吸引し、0.25 %5 mL を加えてトリプシン-EDTA。ストア フラスコ フラスコ表面から細胞を完全にデタッチするのには 5 分の 37 ° C のインキュベーターで</li>。
	<li>、トリプシンを中和するためにフラスコに暖められたメディアの 5 mL を追加します</li>。
	<li>遠心チューブ ミックス トリパン ブルーの 10 μ L と細胞懸濁液の 10 μ L.</li>
	<li>200 x g に 8 分の元の細胞懸濁液を遠心分離</li>
	<li>細胞懸濁液を遠心分離されているが、一方、検定にトリパン ブルー細胞懸濁液の 10 μ L を追加し、セル数を数えるためです</li>。
	<li>以下の遠心分離、細胞ペレットをずらすことがなくメディアを吸引します。1.25 x 10 5 セル/mL の濃度で細胞ペレットを再懸濁します</li>。
	<li>10 cm 回転楕円体成長の間に水和環境を維持するためにシャーレの底に滅菌リン酸緩衝生理食塩水 (PBS) 5 mL を追加します</li>。
	<li>は、シャーレの蓋を反転します。シャーレの蓋の内側の面に細胞懸濁液の 20 μ L を数滴を追加します</li>。
	<li>はすぐに蓋を再び反転、ハングの低下が外れないように注意しながら、皿の底に置きます。 図 1 が正常に形成されたハングの低下を示しています</li>。
	<li>慎重に 37 ° C の定温器皿を配置でき、吊りで成長する回転楕円体 72 h の期間文化をドロップ</li>
</ol> 3。6 日目: 3 D マトリックスへの回転楕円体の埋め込み <ol><li>サンプルの汚染を防ぐために文化フードで以下を実行します</li>。
	<li>最初のフィブリンの層の形成は、血栓のソリューションとして多くの井戸の井戸あたりの 100 μ L を追加する十分なボリュームの血栓ソリューションを作成 (ウェルあたり 1 回転楕円体) 必要です。血栓は、0.1 U/mL 人間 α トロンビン、2 mg/mL フィブリノゲン量 (250 mM HEPES、1500 mM の NaCl、50 mM CaCl 2、pH 7.4) によってバッファーの 10 X HEPES 10% ので構成されます。血栓ボリュームの残りの部分は、純水で構成されています。
	<ol><li>追加トロンビン最後かつ迅速にソリューションを追加 48 ウェル プレートに希望の井戸井戸に追加される前に重合から血栓を防ぐためにします</li>。
	</ol></li> <li>優しくシェイク/タップ フィブリン血栓の混合物は全体をよく、コーティングはようにウェル プレートと重合する底凝固層を許可するように 1 h のためのインキュベーターのウェル プレートを配置。ウェル プレート バブル形成を誘導するのに十分なハードを手ふれしません。単に必要に応じてプレートをロックまで井戸の全体をコーティングします。大量の血栓を使用している場合この手順は必ずしも必要です</li>。
	<li>フィブリン層に回転楕円体を転送します。
	<ol><li>削除もプレートと絞首刑の回転楕円体を含むペトリ皿インキュベーターから文化をドロップし、顕微鏡下で回転楕円体を調べます。文化フードに料理を配置します</li>。
		<li>慎重にアクセスを許可するシャーレのふたを反転吊りする文化にドロップします</li>。
		<li>1 mL の注射器に付いている 21 G 針を使用して慎重に井戸の底をコーティング重合フィブリン層を穴をあけるように注意して、各井戸の中心に逆蓋から一滴を転送します。ドロップを効率的に転送するには、針の中にドロップをゆっくりと引き出します。戻るとすぐに針の内部全体のドロップは、プランジャーを取得を停止します。ドロップをあまりにも遠くに引いて効果的な回転楕円体の転送または後続のイメージングを混乱させることができます空気の泡を引き起こす可能性があります</li>。
		<li>回転楕円体が必要なすべての井戸に正しく転送されるようにするには顕微鏡下でウェル プレートを調べます</li>。
	</ol></li> <li>2 番目のフィブリンの層の形成、最初フィブリン層 (ステップ 3.2) の作成については、上記と同じ準備を使用して別の血栓ソリューションを作成します。慎重に各回転楕円体を含むドロップに 100 μ L のピペットし、再び回転楕円体がそのまま残っているし、井戸の端にプッシュされていないことを確認するには顕微鏡下で調べています</li>。
	<li>インキュベーターにウェル プレートを返し 1 h. の重合に 2 番目の層を許可します。</li>
</ol> 4。6 日目: プロ渡り鳥や抑制因子添加 <ol><li>37 暖かい HDFn メディア ° C</li>
	<li>の汚染を防ぐためにフードで次の手順を実行します</li>。
	<li>評価されるプロ/反 migratory エージェントの適切な濃度を含む各グループのメディアを準備します</li>。
	<li>インキュベーターと文化フードの場所からウェル プレートを削除します</li>。
	<li>回転楕円体群の各ウェルに標準的なメディアの追加 500 μ L、回転楕円体の移行抑制グループの各ウェルに抑制メディアの 500 μ L と移行が強化された回転楕円体グループの各ウェルに pro 渡り鳥メディアの 500 μ L.</li>
	<li>ウェル プレートをインキュベーターに戻ります</li>。
	<li>変化媒体ごとの 48 h.</li>
</ol> 5。日 6-9: 評価の細胞の遊走や増殖 72 h オプションのメディア (0 h) と 24 h に、添加直後に明視野顕微鏡を用いた <ol><li>画像回転楕円体: 各時点で z スタックのイメージを取る場合を決定するために移行は、焦点の主な区域の外の平面で発生しています。我々 の経験では、この問題は発生しません。しかし、これはすべての実験の場合ことを確認すると便利です</li>。
	<li>連続時間の各ポイントで各回転楕円体のセルの枠線をトレースして各回転楕円体の面積のパーセントの増加を分析することにより各ウェルの細胞伸長を決定する使用 ImageJ、&#34; 測定 &#34; 領域を取得します</li>。
</ol>

<ol>
	<li>Chester, D., Brown, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+biophysical+properties+of+provisional+matrix+proteins+in+wound+repair.">The role of biophysical properties of provisional matrix proteins in wound repair.</a> Matrix Biol. , (2016).</li><li>Martin, P., Leibovich, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cells+during+wound+repair:+the+good,+the+bad+and+the+ugly.">Inflammatory cells during wound repair: the good, the bad and the ugly.</a> Trends in Cell Biology. 15 (11), 599-607 (2005).</li><li>Lin, B., Yin, T., Wu, Y. I., Inoue, T., Levchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+chemotaxis+and+contact+inhibition+of+locomotion+determines+exploratory+cell+migration.">Interplay between chemotaxis and contact inhibition of locomotion determines exploratory cell migration.</a> Nat Commun. 6, 6619 (2015).</li><li>Ngalim, S. H., 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=Creating+Adhesive+and+Soluble+Gradients+for+Imaging+Cell+Migration+with+Fluorescence+Microscopy.">Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy.</a> J Vis Exp. (74), (2013).</li><li>Charras, G., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+influences+of+the+extracellular+environment+on+cell+migration.">Physical influences of the extracellular environment on cell migration.</a> Nat Rev. Mol Cell Biol. 15 (12), 813-824 (2014).</li><li>Petrie, R. J., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+Lead+the+Way:+A+Unified+View+of+3D+Cell+Motility.">Fibroblasts Lead the Way: A Unified View of 3D Cell Motility.</a> Trends Cell Biol. 25 (11), 666-674 (2015).</li><li>Petrie, R. J., Gavara, N., Chadwick, R. S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpolarized+signaling+reveals+two+distinct+modes+of+3D+cell+migration.">Nonpolarized signaling reveals two distinct modes of 3D cell migration.</a> J Cell Biol. 197 (3), 439-455 (2012).</li><li>Carlson, M. W., Dong, S., Garlick, J. A., Egles, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Engineered+Models+for+the+Study+of+Cutaneous+Wound-Healing.">Tissue-Engineered Models for the Study of Cutaneous Wound-Healing.</a> Bioengineering Research of Chronic Wounds. , 263-280 (2009).</li><li>Doyle, A. D., Wang, F. W., Matsumoto, K., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+topography+underlies+three-dimensional+fibrillar+cell+migration.">One-dimensional topography underlies three-dimensional fibrillar cell migration.</a> J Cell Bio. 184 (4), 481-490 (2009).</li><li>Fraley, S. I., 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=A+distinctive+role+for+focal+adhesion+proteins+in+three-dimensional+cell+motility.">A distinctive role for focal adhesion proteins in three-dimensional cell motility.</a> Nat Cell Biol. 12 (6), 598-604 (2010).</li><li>Petrie, R. J., Doyle, A. D., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+versus+directionally+persistent+cell+migration.">Random versus directionally persistent cell migration.</a> Nat Rev. Mol Cell Biol. 10 (8), 538-549 (2009).</li><li>Rommerswinkel, N., Niggemann, B., Keil, S., Z&#228;nker, K. S., Dittmar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Cell+Migration+within+a+Three-dimensional+Collagen+Matrix.">Analysis of Cell Migration within a Three-dimensional Collagen Matrix.</a> J Vis Exp. (92), (2014).</li><li>Jonkman, J. E. N., 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=An+introduction+to+the+wound+healing+assay+using+live-cell+microscopy.">An introduction to the wound healing assay using live-cell microscopy.</a> Cell Adh Migr. 8 (5), 440-451 (2014).</li><li>L&#228;mmermann, T., 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=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Chen, Z., Yang, J., Wu, B., Tawil, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Three-Dimensional+Wound+Healing+Model.">A Novel Three-Dimensional Wound Healing Model.</a> J Dev Biol. 2 (4), 198-209 (2014).</li><li>Ross, R., Benditt, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+collagen+formation.">Wound healing and collagen formation.</a> The J Cell Biol. 12 (3), 533-551 (1962).</li><li>Sproul, E. P., Argraves, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytokine+axis+regulates+elastin+formation+and+degradation.">A cytokine axis regulates elastin formation and degradation.</a> Matrix Biol. 32 (2), 86-94 (2013).</li><li>Almine, J. F., Wise, S. G., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin+signaling+in+wound+repair.">Elastin signaling in wound repair.</a> Birth Defects REs C Embryo Today. 96 (3), 248-257 (2012).</li><li>Tracy, L. E., Minasian, R. A., Caterson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+and+Dermal+Fibroblast+Function+in+the+Healing+Wound.">Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound.</a> Adv Wound Care. 5 (3), 119-136 (2016).</li><li>Cox, S., Cole, M., Tawil, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+Human+Dermal+Fibroblasts+in+Three-Dimensional+Fibrin+Clots:+Dependence+on+Fibrinogen+and+Thrombin+Concentration.">Behavior of Human Dermal Fibroblasts in Three-Dimensional Fibrin Clots: Dependence on Fibrinogen and Thrombin Concentration.</a> Tissue Eng. 10 (5-6), 942-954 (2004).</li><li>Brown, A. C., Barker, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin-based+biomaterials:+Modulation+of+macroscopic+properties+through+rational+design+at+the+molecular+level.">Fibrin-based biomaterials: Modulation of macroscopic properties through rational design at the molecular level.</a> Acta Biomaterialia. 10 (4), 1502-1514 (2014).</li><li>Bartosh, T. J., Ylostalo, J. H. <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+anti-inflammatory+mesenchymal+stem/precursor+cells+(MSCs)+through+sphere+formation+using+hanging+drop+culture+technique.">Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging drop culture technique.</a> Curr Protoc Stem Cell Biol. 28, (2014).</li><li>Bainbridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+the+role+of+fibroblasts.">Wound healing and the role of fibroblasts.</a> J Wound Care. 22 (8), 410-412 (2013).</li></ol>

著者が明らかに何もありません。

このプロトコルでは、創傷治癒過程で細胞の移動の検討は、生体外で3D モデルを Ecm を関連付けられているをことができます。プロシージャの適切な実行の重要なステップは、線維芽細胞のスフェロイドの適切な開発2,500 セル/ドロップの初期濃度を与える細胞濃度の準備中に最適化されたフォームに回転楕円体を 72 時間の潜伏期間を確実にできます。細胞の数が少ないを使用する場合...

創傷治癒、組織の整合性の損傷の修復に起因する複雑なプロセスです。このプロセス ステージに分割されます 4 重複止血、炎症、増殖、改造に包まれたそれぞれ規制されている水溶性キューと、細胞マトリックスの相互作用、細胞間コミュニケーションの複雑な組み合わせで。1,2これらの手がかりのオーケストレーションは、創傷治癒など、重要なは、細胞の移動における細胞応答の多数を制御します。3,4細胞の遊走、細胞表現型と細胞外マトリックスの環境の生化学的および生物物理学的特性に依存して非常にダイナミックなプロセスです。5セル連続プローブ-インテグリンを介する経路; を介して細胞外マトリックス環境このプロセスでは、地形、剛性、閉じ込めなどマトリックス プロパティの広い範囲を感知する細胞をことができます。細胞遊走の二次元 (2 D) 解析は、移行がアクチン フィラメント バンドルの世代を通じてリーディング エッジでケラトサイト形成によって駆動されることを示します。前縁、後縁の収縮と収縮を駆動アクトミオシンとの組み合わせで焦点接着斑の形成は、セルを突き動かします。6三次元 (3 D) 細胞の移行は現在よくわかっていない, しかし、最近の研究は示す 3 D 移行は著しく異なること、船首に 2 D のメカニズムを説明し、変化をもたらすメカニズムによって駆動される可能性が高い。たとえば、ピートリーらによる最近の研究。ECM のプロパティ、に応じて 3 D マトリックス内のセルと切り替えることが lamellipodium 移行のメカニズムを駆動 lobopodium を示した。7細胞の遊走は創傷治癒応答の重要な要素は、細胞遊走の 3 D モデルは創傷治癒過程における様々 な刺激に対する生理反応を理解すること重要です。3 D マトリックスの移行とは大きく異なる 2D サーフェス上セル回遊行動を示されている、創傷治癒過程、老舗のスクラッチ アッセイを含む中細胞遊走の最新の in vitroモデル利用 2 D単層培養。5,6,7,8,9,10,11,12,13,14より最近開発されたアッセイ 3 D マトリックスを利用してきたが、まだこうして本当に細胞環境の立体感を再現する能力を制限、マトリックス上に単層細胞の培養体内。15
記載方法の目的は、応用刺激に関連付けられている移行反応のより堅牢な理解を得るために細胞遊走 2D サーフェスではなく生理学的関連性の高い 3 D モデルを勉強することです。このメソッドは、アクティブ化または細胞の移動に関連付けられている経路を阻害する要因の存在下で 3 D フィブリン血栓マトリックス内の細胞の移動の評価できます。創傷治癒の初期段階で重要な役割フィブリンによるこのメソッドの選ばれたフィブリン マトリックス次の傷害、フィブリン血栓が血液の損失を食い止めるため創傷環境で形成される組織の修復と改造の間に初期の細胞浸潤の足場として提供しています。2,16,17,18,19,20はまた、フィブリンが外科シーラントとして臨床的に使用され、シーラントの組成は細胞遊走と究極の成果を癒しに縛られています。コックスらによる前の調査。さまざまなフィブリンと、フィブリンの最適化を目的としてトロンビン濃度フィブリン血栓を線維芽細胞の移行を検討しました。これらの研究では、プラスチック製の食器にフィブリン血栓から細胞の移動の定量化を行った。20ここで 3 D フィブリン環境内で細胞の移動の定量化できる方法を紹介します。具体的にはこのプロトコルで記述されているメソッドは、フィブリンを使用して、このモデルはコラーゲンや他の 3 D の行列など、必要に応じて代替マトリックス材料を使用する簡単に変更できます。さらに、このアッセイで線維芽細胞スフェロイドの使用は, 傷ベッドに線維芽細胞の移行が最も重要な細胞外マトリックスの合成と組織修復/改造するので.ただし、修理中にプロセスの好中球はマクロファージが続く傷のベッドに移行する最初のセルの種類です。線維芽細胞は、約 48 時間後に外傷、創傷治癒過程の増殖期の初めに傷環境に侵入を開始します。19のこのアッセイは、好中球、マクロファージ、フィブリン血栓構造の変化によるこれらの細胞型の移行の影響を査定する他の細胞型などに簡単に修正できます。21
このアッセイを実装するには、最初に、線維芽細胞スフェロイド形成幹細胞培養のために紹介した方法の変更を使用しています。22は線維芽細胞スフェロイド、3 D フィブリン マトリックスに転送後、伸長する簡単に監視し、数日間定量化します。このプロトコルは、フィブリン マトリックス内 3 D 細胞スフェロイドを埋め込むことによって考慮に生理学的システムの 3 D は中にもたらされるモデル回遊行動に細胞膜の 2次元成長表面を使用して、関連性の制限を避けるため、まだ厳しく体外環境における細胞挙動の評価が可能。

<table border="0" cellpadding="0" cellspacing="0" width="692">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Materials</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Dulbecco&#39;s Modified Eagle&#39;s Medium, 4.5 g/L Glucose, w/ Sodium Pyruvate, w/out L-Glutamine</td>
			<td style="width:91px;">VWR</td>
			<td>VWRL0148-0500</td>
			<td style="width:217px;">DMEM already containing L-glutamine can also be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">100mm Tissue Culture Dish, Non-Treated, Sterilized, Non-Pyrogenic</td>
			<td style="width:91px;">VWR</td>
			<td>10861-594</td>
			<td style="width:217px;">Dishes of any size can be used for hanging drop culture&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Fetal Bovine Serum from USDA approved countries, heat inactivated, sterile-filtered, cell culture tested</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>12306C-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">L-glutamine</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td>ICN1680149</td>
			<td style="width:217px;">Not needed if using DMEM that already contains L-glutamine&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Penicillin-Streptomycin Solution stabilized, sterile-filtered, with 10,000 units penicillin and 10 mg streptomycin/mL</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Human Dermal Fibroblasts, neonatal</td>
			<td style="width:91px;">Thermo Fisher</td>
			<td>C0045C</td>
			<td style="width:217px;">Can be replaced with other cell type of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">21 G x 1 1/2&#39; needle</td>
			<td style="width:91px;">BD&#160;</td>
			<td>305167</td>
			<td>22 G x 1 1/2 needles will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">1 mL syringe</td>
			<td style="width:91px;">VWR</td>
			<td>89174-491</td>
			<td>Syringes of any volume can be used</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Cell Culture Multiwell Plates, Polystyrene, Greiner Bio-One (Individially Wrapped) (48 wells)</td>
			<td style="width:91px;">VWR</td>
			<td>82051-004&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Fibrinogen - Plasminogen, von Willebrand Factor and Fibronectin Depleted</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>FIB 3</td>
			<td style="width:217px;">Can be replaced with matrix protein of interest</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Alpha Thrombin</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>HT 1002a</td>
			<td style="width:217px;">May not be necessary depending on matrix protein of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Equipment</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSL2 cell culture hood</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell culture incubator</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Inverted microscope with 10X objective</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Centrifuge compatibile with 15 mL tubes</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing cell migration within three-dimensional in vitro wound environments

現在のほとんどの生体外モデルは傷の治癒、老舗スクラッチ アッセイなど、2次元の表面上のセルの移行および創傷閉鎖の勉強を含みます。しかし、どの生体内で創傷治癒が行われる生理学的な環境は二次元ではなく三次元です。セルの動作が大幅に異なることはますます明確になっている二次元三次元環境対したがって、傷の閉鎖の細胞移行挙動を勉強してもっと生理学的関連性の in vitroモデルの必要性があります。記載方法より以前に確立された二次元スクラッチ アッセイより生理学的条件を反映した三次元モデルの細胞移動の調査のためことができます。このモデルの目的は、プロまたは反 migratory 因子存在下でフィブリンのマトリックスに埋め込まれた回転楕円体ボディから細胞遊走の検討を通して細胞伸長を評価することです。このメソッドを使用して、三次元マトリックスの回転楕円体ボディから細胞伸長を観察し、明視野顕微鏡および回転楕円体の本体領域の解析による時間の経過とともに容易に定量化できないです。セル移動に及ぼすプロ渡り鳥あるいは阻害要因は、このシステムでは評価できます。このメソッドは、関連付けられている三次元傷行列の in vitro細胞移動分析の単純なメソッドを持つ研究者を提供します、このように体外の関連性を高める細胞研究生体内で動物を使用する前にモデル。

正常にプロおよび反渡り鳥エージェントの体外線維芽細胞に及ぼす影響を評価する回転楕円体文化を利用できます。 72 h (図 1) の期間にわたってドロップ文化をぶら下げを介して 3 D 線維芽細胞スフェロイドを形成できます。培養期間が過ぎると、回転楕円体は、血栓のマトリックスに埋め込まれ、対物レンズ (図 2...

Watch this Scientific Journal Video about Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments at JoVE.com

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

このプロトコルの目的は、三次元フィブリン マトリックス内の細胞の移動に及ぼすプロおよび反 migratory 因子を評価するためです。

三次元培養細胞遊走を特徴付ける傷の環境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

characterizing-cell-migration-within-three-dimensional-vitro-wound

Research

JoVE Journal

Bioengineering

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

7.6K ビュー.  North Carolina State University and The University of North Carolina - Chapel Hill. このプロトコルの目的は、三次元フィブリン マトリックス内の細胞の移動に及ぼすプロおよび反 migratory 因子を評価するためです。

ビデオ: Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三次元光学分解能光音響顕微鏡

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

生物工学

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2Dと3D環境での神経前駆細胞の電界制御監督移行

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

マイクロインジェクションアッセイをキズや<em&gt;インビボ</em&gt;表皮の局在は、応答記者傷<em&gt;ショウジョウバエ</em&gt;胚。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

の調製と分析<em&gt;インビトロ</em&gt;三次元乳癌サロゲート

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三次元ペプチド足場で哺乳動物の細胞を培養

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

ケラチン生成細胞増殖の 2 次元と 3 次元の評価タイプ I コラーゲン基板

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...