Name: a novel in vitro wound healing assay to evaluate cell migration
Uploaded: 2018-03-17T13:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

この作品は、ローマ ・ ラ ・ サピエンツァ大学とイタリアの嚢胞性線維症研究財団 (プロジェクト FFC #11/2014 年シエナ、ソンドリオ Valchiavenna、Cerea Il ソリーゾ ・ ディ ・ ジェニー、パヴィア FFC 代表団によって採用) からの資金によって支えられました。この仕事の一部はまたによって FILAS グラント Prot. FILAS-RU-2014-1020 をサポートされていました。
気管支の上皮細胞を提供するため博士ロレッタ フェレーラ (U.O.C. Genetica ・ メディカ、Istituto Giannina ガスリーニ、ジェノバ、イタリア) に感謝しております。

1. セル準備
<ol><li>10% 牛胎児血清 (FBS)、抗生物質 (ペニシリンとストレプトマイシンの 0.1 mg/mL) およびピューロマイシン プラス 2.5 x 106 10 mL の最小必須培地 (MEM) 2 mM グルタミン (MEMg) を添加した細胞を播く (0.5 μ G/ml の選択と維持のため、行のセル) T75 フラスコ。2 日間の 37 ° C、5% CO2でフラスコを孵化させなさい。実験を始める前に、倒立顕微鏡下で細胞の合流点を確認します。 注: 実験用セルは、レンチウイルスベクターをピューロマイシン耐性で導入した不死化ひと気管支上皮細胞です。安定機能 CFTR14,15を表します。</li>
	<li>セルの合流は 90-100% に達したら、フラスコから培地を吸引し、生物学的安全キャビネット クラス II の下で廃棄ボトルにそれを破棄します。カルシウムとマグネシウム (CMF PBS) なし生理食塩水リン酸緩衝の ml の 6 セルを洗います。優しく、フラスコを手動でロックし、CMF PBS を破棄します。 注: ピペットで細胞膜を触れないように気をつけてください。</li>
	<li>CMF PBS の 10 mL を追加し、10 分間の 37 ° C および 5% の CO2でフラスコを孵化させなさい。</li>
	<li>CMF PBS を吸引し、それを破棄します。トリプシン/EDTA の 2 mL をフラスコに追加します。</li>
	<li>完全に細胞が顕微鏡の下で目に見えて戸建まで 10 分の CO2を 37 ° C、5% でフラスコをインキュベート細胞をコートしソリューションを許可するフラスコみ合います。 注: インキュベーション時間の終わりに、セルが丸いとプラスチック表面にもアタッチされていないに表示されます。まあ戸ないセルは、手動撹拌が必要かもしれません。</li>
	<li>MEMg プラス 10% の 10 mL を加えてトリプシンを不活化し、フラスコの底を洗浄することにより細胞を収集する政府短期証券。50 mL のコニカル チューブにボリュームを転送します。</li>
	<li>80 x gで 5 分間チューブを遠心します。</li>
	<li>上清を吸引し、再 MEMg プラス 10% の 6 mL のセルを中断政府短期証券。ピペット繰り返し任意の塊を破る。</li>
	<li>マイクロ ピペットで細胞を懸濁液の 10 μ L を取り出して、以前 Burker またはノイバウアー室の上に置くカバー ガラスの下でボリュームを挿入します。</li>
	<li>セル数をカウントします。</li>
</ol>2. 細胞文化の播種を挿入します。
<ol><li>12 ウェル プレートの各ウェル、滅菌ピンセット (図 1) で文化挿入を転送します。板の表面にそれらを修正するために挿入のエッジに沿って押すようにピンセットを使用します。 注: 挿入により接着性粘着性の下側があります。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56825/56825fig1.jpg"> 図 1: 12 ウェル プレートのウェルに正しく入れて、シリコーン文化挿入の略図。<a href="//cloudflare.jove.com/files/ftp_upload/56825/56825fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol start="2"><li>正しく MEMg プラス 10% の細胞懸濁液を希釈 FBS。細胞懸濁液 (約 3.5x104セル/室) の 70 μ L で、挿入の各区画を入力します。 注: 各区画に適用されるセルの密度は、細胞の種類によって異なります。24 h 内で合流の細胞密度を使用することをお勧めします。</li>
	<li>顕微鏡で細胞挿入コンパートメントから漏れていないと 37 ° C、5% CO2で 24 時間 12 ウェル プレートを孵化させなさいことを確認します。</li>
</ol>3. 擬似創傷治癒アッセイ
<ol><li>インキュベーション後、コンフルエントの細胞が形成されていることを確認を倒立顕微鏡下で細胞を可視化します。</li>
	<li>再 1 ml MEMg のテスト混合物 (例えばアンプ) を中断します。 注: 準備新鮮なアンプ希釈原液-20 ° C で保存から始まって</li>
	<li>滅菌ピンセットでチップを慎重に取り外します。細胞膜を壊さないように注意してください。転送吸収紙の上に挿入します。 注: 同じ挿入を再利用するには、滅菌に少なくとも 3 時間の 70% エタノールで。その後にそれらを捨てることをお勧めします。</li>
	<li>非付着性のセルを削除するには、マイクロ ピペットを使用してあたり MEMg の 1 mL を追加します。プレートを閉じ、そっとそれをロックします。 注: は、混乱を避けるために細胞膜上に直接メディアを追加しないでください。</li>
	<li>培地を吸引し、ウェルあたり MEMg の 1 mL と置き換えます。プレートを閉じ、ビデオ カメラを装備して、4 倍の倍率で倒立顕微鏡下で (挿入によって作成された) 携帯無料ギャップを可視化します。画像時 0 (T0) を取得し、.jpg 形式で保存します。</li>
	<li>井戸からメディアを取り出して、1 mL の PBS で洗浄し、それを破棄します。</li>
	<li>井戸に (3.2 の時点で準備) テスト混合物を追加します。無処理試料 MEMg の 1 mL を追加します。37 ° C と 5% の CO2でプレートを孵化させなさい。</li>
	<li>15、20、および 24 時間治療後、4 倍の倍率の顕微鏡下で細胞の移動を観察し、画像を取得します。 注: この手順では、T0 は同じエリアの画像をキャプチャする試み。イメージがキャプチャされます時間間隔は、セルの移動速度によって決まります。</li>
	<li>いくつかの選択的阻害薬の効果を研究、すなわちAG1478、細胞移動への挿入を削除する前に各挿入のコンパートメントから媒体を吸引します。<ol>
<li>新鮮な MEMg と各コンパートメントを洗浄し、AG1478 を添加した MEMg の 70 μ L でそれを埋めます。</li>
		<li>37 ° C および 5% CO2で潜伏の 30 分後 3.3 ポイントから説明するように進みます。 注意: 洗浄のステップおよび細胞特異的阻害剤単分子膜の前処理中に挿入を削除しないように注意します。</li>
	</ol>
</li>
</ol>4. 画像解析
<ol><li>実験が完了したら、さまざまな実験的グループの最も代表的なサンプルの画像を選択し、T0、T15、T20、T24 h で 1 つの画像を含む zip ファイルを作成します。 注: 単一の画像はすべてサンプルを選択した時間間隔で撮影されます。少なくとも 3 回を繰り返し、それぞれの実験の実行をトリプリケートします。最後に、すべての実験的グループ、("a"、"b"、"c"、等、それぞれの独立した実験から派生する) 3 つの画像の最小の各時点で分析されます。</li>
	<li>自動的に電子メールで選択した時点で (パーセンテージ) 細胞に覆われたとスクラッチ領域の実験的データを含むスプレッドシートの概要ファイルを提供する画像解析ソフトに zip ファイルをアップロードします。 注: リーディング エッジとギャップ領域の認識は (目的で画像の明るさが大幅に変更された点を識別する) エッジ検出法に基づいて。</li>
	<li>データを保存し、それらを収集します。時間 0 で平均値に対するすべてのデータを正規化します。各時点で相対的標準誤差 (SEM) すべて複製の正規化されたデータの平均値を計算します。双方向分散分析 (ANOVA) を使用して、適切な統計解析ソフトウェアの統計分析を実行します。ペプチド投与群と異なる時間間隔で制御グループの違いと見なされる統計的に重要なpの < 0.05。</li>
	<li>グラフに、選択した時間間隔すべてサンプル グループ対の細胞で覆われた領域のパーセントを示すヒストグラムとして得られたデータをプロットします。</li>
</ol>

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著者がある何も開示するには

細胞遊走は、創傷治癒、萌芽期の開発、がんの転移など多くの生理学的および病理学的イベントに欠かせないプロセスです。細胞移行生体外で勉強する基本的な手順を含む: (iii) 傷までのさまざまな時間間隔での画像キャプチャ閉鎖に達する (i) 細胞膜、(ii) 細胞の合流の層で擬似傷の生産の作成、および (iv) 選択したセルの移行速度を定量化するために, 動画像の解析。
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動物の創傷治癒過程がけが1後の整合性と組織層の正常な機能に再確立する基本的なプロセスであることを主として知られています。物理・化学的侮辱から防護壁を形成上皮表面 (例えば、呼吸器、皮膚、消化管) の外部環境にさらされているにもかかわらず、傷の形成が発生しやすく、特に後手術または微生物感染症2。例として、嚢胞性線維症 (CF) 患者において日和見細菌菌緑膿菌、によって肺組織の植民地化とそれに伴う呼吸不全3、気道上皮の損傷につながる 4。創傷治癒は、負傷した組織5通常の建築を復元する複雑なホスト修理メカニズムです。上皮形成、血管新生、コラーゲン産生と細胞分化6,7,8 組織の改造現象を包括的な再生期間続いて初期の炎症によって特徴づけられる.上皮の完全性を確保するため、微生物の増殖を制御するため、すべての生きている有機体は抗菌ペプチド (アンペア)9,10を含む防衛分子を生成します。創傷治癒過程シミュレートの in vitro細胞残屑および異なる種類の細胞間の複雑な相互作用の欠乏のために非常に困難です。ただし、ペプチドの生体外で能力を刺激することによって擬似傷の閉鎖を加速する上皮細胞の移行は侵害された上皮を治癒する能力を示す。確かに、細胞遊走は創傷治癒にレート制限イベントと改良された創傷治癒のためのターゲット治療に役立つ細胞の移動に影響を与える要因を勉強します。
ここでは、再現性の高い実験的試金はセル移行体外を評価する特殊シリコン文化挿入に基づいて提供しています。それは、コンフルエントの細胞膜上に 500 μ m ギャップ (擬似傷) の作成に基づいています。無料のセル領域に移行する、新しい細胞間の接触を形成、人工の「負傷者」のフィールドの端にあるセルが開始されます。文化の挿入は、高速治癒促進実験のための新しいツールを表します。500 μ m の壁で区切られた 2 つの貯水池が提供され、12 ウェル プレートの井戸、3 cm 皿プレートにそれら正しく配置ことができます。挿入の各コンパートメントの細胞懸濁液を充填セル挿入の除去は (分離壁と同じ幅) 約 500 μ m のきれいな無細胞のギャップを生む一方、confluence (コンフルエンス) まで各指定地域で成長することができます。試験化合物を添加した適切な細胞培養液を皿に追加できるプレート/ウェル。その後、ギャップの閉鎖は、好ましくは 1 つの画像取り込みビデオ カメラ搭載、倒立顕微鏡下で異なる時間間隔で視覚化できます。最後に、web ベースの自動画像解析プログラムによってセル面積の変化の測定には、創傷閉鎖と細胞移動の速度の定量化ができます。全体的にみて、この方法は、スクラッチは滅菌した針でコンフルエントの細胞膜を切開によってなされる手動でまたはピペット チップ11クラシックのアッセイに関して一歩前進です。確かに、最後の手順は、皿のプラスチック底を破壊できるプレート/まあ、表面コーティング、しわを作成します。さらに、高針の先端に研究者によって適用される圧力にもよりますので、「負傷者」の領域には、ギャップの全体の長さに沿って明確に定義された幅がありません。さらに、剥がれ細胞が、ゼロの端の生活および死んだ細胞の塊を形成できます。また、「負傷者」の領域に生きている細胞の広がりは、12非再現性のある結果を生成する細胞移動の速度と干渉することができます。
さらに、スクラッチのイメージ解析プラットフォームのおかげでユーザー急速に受信できる (数分) 追加のソフトウェアを取得する必要なく選択したセルの回遊行動の定量的なデータ。このプラットフォームは、低 (~ 5 X) の位相コントラスト顕微鏡画像、中規模 (~ 10 X)、および (~ 20 倍) の高倍率を分析が可能です。セルの速度だけでなく、細胞に覆われたエリアとスクラッチ領域の割合を示すサマリー ファイルを生成する (*.jpg、*.jpeg、*.jp2, *.png, *.gif, *.tiff 形式) で画像の zip ファイルをアップロードした後、分析は自動的に行なわれます移行は、異なる時間間隔で。
カエル皮膚のアンプ誘導体、すなわちEsc(1-21) とそのジアステレオマー Esc(1-21)-1 c13、および機能 CF 膜コンダクタンス レギュレータ (CFTR)14,15を表現する気管支細胞ラインを使用して、この作業でペプチドによる細胞移行 (無処理) サンプルとの比較の例を示します。気道上皮、CFTR が肺機能を維持するために重要な役割を果たすことに注意してください、修理16を傷します。さらに、による上皮成長因子受容体 (EGFR)、前述のペプチドによる気管支の細胞の移動を含む EGFR12の活性化の証拠の選択的阻害剤 (例えばAG1478)17 18の報告します。
この手順の目標は文化そのようなシリコンを挿入の使用状況が正確に付着性のセル (例えば、気管支上皮細胞) と分子の移行を判断する迅速かつ簡単にアクセスできるアッセイをどのように表現するかを要約すると、このようなイベントを制御するメカニズムです。

<table><tbody><tr><td>Minimum essential medium (MEM)&amp;nbsp;</td><td>Euroclone</td><td>ECB2071L</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Glutamine</td><td>Euroclone</td><td>ECB3000D</td><td></td></tr><tr><td>Heat inactivated Fetal Bovine Serum (FBS)</td><td>Euroclone</td><td>ECS0180DH&amp;nbsp;</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>Euroclone</td><td>ECB3001D</td><td></td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P8833</td><td></td></tr><tr><td>Trypsin/EDTA 1X in PBS</td><td>Euroclone</td><td>ECB3052D</td><td>Warm at room temperature before use</td></tr><tr><td>DPBS without calcium and magnesium (CMF-PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>DPBS with calcium and magnesium (PBS)</td><td>Sigma-Aldrich</td><td>D8662</td><td></td></tr><tr><td>Ibidi Culture-Insert 2 well</td><td>Ibidi&amp;nbsp;</td><td>80209</td><td></td></tr><tr><td>Wimasis Image Analysis</td><td>Ibidi&amp;nbsp;</td><td>30002</td><td></td></tr><tr><td>PRISM software&amp;nbsp;</td><td>GraphPad</td><td>version 6.0</td><td></td></tr></tbody></table>

a novel in vitro wound healing assay to evaluate cell migration

この作業の目的は、抗菌ペプチド (アンペア)、細胞の移動を刺激するなどのいくつかの免疫調節分子の能力を評価する新しい方法を提示することです。重要なは、細胞遊走、傷害後の整合性と組織層の正常な機能に再確立する創傷治癒プロセスの間にレート制限イベントです。手動で作られた単層細胞傷に基づいている古典的な分析上のこのメソッドの利点は、特殊シリコン文化の使用は明確に定義された幅 (500 μ m と無細胞の擬似巻きフィールドを作成する 2 つのコンパートメントを提供するを挿入します).さらに、自動画像解析プラットフォーム、ため急速に創傷閉鎖と細胞移動の速度で定量的なデータを得ることが可能です。正確には、気管支上皮細胞の移動に 2 つのカエルの皮アンプの効果が表示されます。さらに、これらの細胞特異的阻害剤での前処理は分子機構などのイベントに関する情報を提供します。

このプロトコルは創傷治癒機能 CFTR を表現する気管支上皮細胞に誘発される細胞の移行活動の面で Esc(1-21) と Esc(1-21)-1 c の効果を決定するために使用されました。このアッセイで文化挿入された 12 ウェル プレートの井戸に置かれ、各コンパートメントは 35,000 MEMg 10% を添加した細胞が播種された FBS。セルその後 24 時間以内の完全な合流点に達した、500 μ m ギャップ?...

Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

ここでは、ペプチドの気管支上皮細胞の移動に及ぼす影響を評価するためのプロトコルを提案する.このメソッドは、セル移行および創傷閉鎖の速度に定量的なデータの迅速かつ再現性の高い国又できます。

新規In Vitro細胞移動を評価する癒しのアッセイを傷

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

a-novel-in-vitro-wound-healing-assay-to-evaluate-cell-migration

Research

JoVE Journal

Immunology and Infection

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

21.7K ビュー.  Sapienza University of Rome. ここでは、ペプチドの気管支上皮細胞の移動に及ぼす影響を評価するためのプロトコルを提案する.このメソッドは、セル移行および創傷閉鎖の速度に定量的なデータの迅速かつ再現性の高い国又できます。

ビデオ: A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

有害化学物質への曝露後の内皮細胞の遊走の評価

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

発生生物学

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

細胞浸潤および移行プロセスの評価: ビデオ顕微鏡ベースのスクラッチの比較創傷アッセイと boyden 教授区域の試金

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

がん研究

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

96ウェルプレートに細胞遊走/創傷治癒を行う装置

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

体外皮膚細胞移動へのヒ素の効果を発揮するアッセイをスクラッチします。

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

環境学

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 ...

生物工学

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

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 ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

顕微鏡法によるIn Vitro創傷が治癒過程における上皮移行の評価

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

医学

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

インビトロでの細胞遊走および浸潤アッセイにおける

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

生物学

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

インビトロおよび創傷治癒の生体内分析のためのスクラッチ移行アッセイとドーサルスキンフォールドチャンバー

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.