Name: a device for performing cell migration/wound healing in a 96-well plate
Uploaded: 2017-03-07T15:00:00.000+00:00
Duration: 5 min 46 s
Description: Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

著者は、このwounderのプロトタイプを製造する際に氏タムポー・レオン氏ウォンチー金武とその技術的なスキルのための科学学部ワークショップの技術スタッフ、香港バプティスト大学、アドバイスに感謝したいと思います。

1. Wounderのパーツの概要（図1） <ol><li>等間隔に創傷のピンを固定することにより、ピンホルダーを準備します。ピペットチップを持ち、上下に調節可能な創傷ピンを移動させることにより、先端の高さを調整します。 </li><li> 96ウェル培養プレートの異なるブランドに調節可能なガイドバーを取り付け、創傷領域が固定位置にあることを確認してください。 </li><li>六角ネジを締めて、調整可能な創傷固定してください。両端の六角キャップを締めて位置の案内棒を固定します。 </li></ol> 2.ピンホルダーの幅を設定する（図2） <ol><li> M5六角レンチで六角キャップを緩めます。 </li><li>ガイドバーは、96ウェル培養プレートの幅に完全に収まるように、ピンホルダーの両側に調節リングの適切な数を挿入します。 注：コーニングといわきプレート大リングの1対の小さなリングの1組を必要とします。ファルコンとヌンクPLATEは、大きなリングの1ペアが必要。 </li><li>六角を締め案内バーを固定するキャップ。 </li></ol> 3.ガイディングバーを調整する（図3） <ol><li>滅菌10-μLプラスチックピペットチップで負傷ピンを取り付けます。 </li><li> M5六角レンチで六角キャップを緩めます。 </li><li> wounderを保持し、96ウェル培養プレートの1列に創傷ピンを浸します。 </li><li>ヒントのすべてがかろうじてウェルの底に触れるまで案内するバーの高さを調整します。 </li><li>六角キャップを締めます。 wounderは現在、完全に培養プレートに適合し、ピンをウェルの中央によく配置されていることをことを確認してください。 </li></ol>ピンの4キャリブレーション<ol><li>平らな無菌面に垂直なwounder（ すなわちシャーレ）を持ち、M3六角レンチですべての六角ネジを緩めます。 </li><li>ヒントのすべてが均等に平らな滅菌表面に接触するまでwounderをタップします。&lt;/李&gt; <li>位置にピンをロックするために、再び六角ネジを締めます。 </li><li>平らな無菌の表面を軽くwounderをタップすることで、各チップの均一性を確認してください。 </li></ol> 5.スクラッチ細胞単層（図4） <ol><li> 0.1％ゼラチンでコーティングした96ウェル細胞培養プレートにヒト臍帯血管内皮細胞を播種します。 20％熱不活化ウシ胎児血清、1％ペニシリン/ストレプトマイシン0.09グラム/ Lのヘパリンを添加した培地199中で培養細胞。一晩使用される前に、37ºCで5％二酸化炭素を含む加湿インキュベーター内で細胞を維持します。細胞が負傷する前に、100％の密集度に到達する必要があります。 </li><li>一番左（または右端）ウェル培養プレートの同じ列内の各側の創傷のヒントを配置します。ウェルの反対側に渡ってwounderをスライドさせます。創傷先端がウェルの底に触れていることを確認してください。 </li><li>すべての列のスクラッチ（ステップ5.2）を繰り返します。 </li><li>負傷後、トンを廃棄各ウェル中の培地彼と試験化合物を含む新鮮な培地と交換してください。 </li></ol> 6.データ収集と画像解析<ol><li>画像デジタルカメラ（ 例えば 10倍の対物レンズ）を備えた低倍率の顕微鏡を用いて細胞単層上の機械的創傷と全体だけでなく、すぐに（T = 0時間）創傷後。 </li><li>必要であれば、試験化合物を追加して、所望の時間インキュベートします。創傷領域と画像全体をよく再び希望のインキュベーション時間後（T =Δhを）。 </li><li>そのようなImageJのような画像解析ソフトウェアを（使用して<a href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> ）、フリーハンドツールを使用して、画像上の創傷領域を測定します。 </li><li>創傷閉鎖の割合を計算します。 <img alt="方程式" src="/files/ftp_upload/55411/55411eq1.jpg" /></li></ol>

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</li><li>Chen, Y. C., 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=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci Rep. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. 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+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. Y., 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=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

著者は、彼らが競合する金融利害関係を持たないことを宣言します。

私たちの細胞wounderは、従来の細胞遊走アッセイの問題を解決するにはいくつかのユニークな特徴を持っています。 8チャンネル機械細胞wounderは、オートクレーブにより滅菌することができる長寿命で高品位のステンレス鋼（スチール304）で構成されています。市販の96ウェル培養プレートのほとんど全ての商業ブランドがあるため、調整可能なガイドバーのこの機械的な細胞wounderと共に使用...

細胞遊走は、胚発生、神経発生、血管新生、創傷治癒、粘膜修復、正常な発達と病気の状況1下上皮-間葉遷移などの細胞プロセスにおいて重要な役割を果たしています。これは、シグナル伝達分子間相互作用、細胞極性、細胞骨格の再編成、マトリックスリモデリング、膜の突起と動的細胞間接着の変調2を含む多数の間および細胞内のイベントの調整を必要とする複雑なプロセスです。細胞遊走を研究することによって、細胞運動およびその関連する生化学的経路をもたらす化学物質または生体分子の発見および検証を決定することができます。この基本的なプロセスは、このような疾患の治療および薬物標的化のような種々の用途のためのプロキシとして有利に使用することができます。 創傷アッセイは、細胞遊走アッセイ3の一つです。ここでは、細胞の個々のサンプルは、部分的に、細胞が領域をカバーするように移動する露出領域を生成するために機械的手段によって除去されます。特定の時点で回収率が監視されます。多数のサンプルを取り扱う場合、そのようなサンプル内の実験的なコストと交差汚染などの問題は、問題となる可能性があります。多くの商用ツールおよびアッセイは、細胞遊走4、5、6を研究するために利用可能であるが、それらの多くは、高価で精巧な装置を必要とし、改善が依然として必要とされています。これらの理由のために、8チャンネル機械細胞wounder（ 図1）が開発されています。 我々の8チャンネル機械細胞wounderは、上記課題を解決するにいくつかのユニークな特徴を組み込んでいます。これは、96ウェル培養プレートフォーマットでの細胞遊走/創傷アッセイを実行するための柔軟性を提供します。調整可能な区idingバーのデザインは、スクラッチ領域は、各ウェルの中央の位置にあることを保証します。 wounder培養プレートの様々なブランドのために適用可能であるように、また、ガイドバーの高さを調整することができます。最後に、調整可能な創傷ピンの設計は、同時かつ再現性の創傷7、8を達成するために、培養皿の表面にピペットチップであっても接触を可能にします。

<table><tbody><tr><td>96-well cell culture plate</td><td>Nunc</td><td>167008</td><td>Other brands of 96-well cell culture plate can also be used</td></tr><tr><td>P10 pipette tips</td><td>Axygen</td><td>301-03-051</td><td>Short P10 pipette tip is more easy to create a clear wound</td></tr><tr><td>Wounder</td><td>R&amp;amp;P Technology Limited</td><td></td><td></td></tr><tr><td>Medium 199</td><td>Sigma</td><td>M2520-1L</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>26140079</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma</td><td>H3393</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma</td><td>G9391</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr></tbody></table>

a device for performing cell migration/wound healing in a 96-well plate

細胞遊走は/アッセイを負傷して細胞遊走および血管新生および腫瘍転移などの他の生物学的プロセスを研究するために一般的に用いられる方法です。このアッセイでは、細胞は、コンフルエントな単層を形成するために増殖させ、機械的な傷は、デバイスとのスクラッチによって作成されます。次いで、露出領域に向かって細胞の移動速度は、イメージングによってモニターすることができます。当社の8チャンネル機械wounderは、細胞遊走アッセイに関連する問題の多くに対処するように設計されています。まず、私たちのwounderは簡単にオートクレーブ処理によって、あるいは共通の消毒剤で滅菌することができます。第二に、個別の調整可能なピンでもシャープで再現性の創傷を作成することができるように、細胞培養プレートとの接触を可能にします。第三に、wounderの両側の案内バーは、各ウェル中に一貫性のある創傷の位置を確認してください。さらにwounderのより良いハンドリングを提供できるだけでなく、クロスcontaminatを最小限に抑えるために負傷のための使い捨てのプラスチック製ピペットチップの使用イオン。結論として、我々の細胞wounderは、標準的な96ウェル培養プレートを用いて細胞遊走アッセイを行うためのユーザーフレンドリーで再現可能なデバイスと研究者に提供することができます。

この8チャンネル機械wounderは、細胞遊走アッセイを実行するために、細胞単層を傷つけるように構成されています。それは、特別な訓練なし分未満で96ウェルプレート中の細胞引っ掻きアッセイを行うことができるユーザーフレンドリーなデバイスです。このwounderは600μm程度の均一な幅とし、シャープなエッジで細胞単層上の創傷領域を導入することができる（ <strong...

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

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

ここでは、96ウェル細胞培養プレートに半ハイスループット細胞遊走アッセイを実行するためのプロトコルを提示します。このプロトコルは、細胞単層上で一貫性のあるスクラッチ傷を作成するには、高速、シンプルかつ経済的な方法です。

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 ...

a-device-for-performing-cell-migrationwound-healing-in-a-96-well-plate

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.5K ビュー.  Hong Kong Baptist University. ここでは、96ウェル細胞培養プレートに半ハイスループット細胞遊走アッセイを実行するためのプロトコルを提示します。このプロトコルは、細胞単層上で一貫性のあるスクラッチ傷を作成するには、高速、シンプルかつ経済的な方法です。 

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

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

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

線維芽細胞移動の調節にMRP4含有高分子複合体を研究するための方法

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

発生生物学

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

自動生細胞固体撮像素子を使用して単純な移行/侵略ワークフロー

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

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 ...

生物工学

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

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 stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

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 ...

免疫・感染学

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 ...

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

粒状ハイドロゲル生体材料内および粒状ハイドロゲル生体材料内への三次元細胞移動の定量化

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

簡単な2Dおよび3D細胞培養を可能にするように設計された革新的な3Dプリントインサート

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...