著者は大幅キュリー研究所（CNRS UMR144）でPICT IBiSAプラットフォームを認める。この作品は、からの補助金によって賄われていた。AM.LD欧州研究評議会（Strapacemi 243103）、協会国立はMPと午前·ラ·ルシェルシュ（ANR-09-PIRI-0027-PCVI）、InnaBiosanté基盤（Micemico）を注ぐ。 AM.LD.にLDとERC Strapacemi若手研究助成金

重要な注意：このプロトコルは、希望するマイクロチャネルのための形状を含む、型が既に行われていることを前提としています。金型の調製に関するさらなる情報は既に10を出版されました。このプロトコルはまた、骨髄DCは文化が知られていることを前提としています。 1。チップ製造<ol><li>プラスチックカップ中の重量比10:1でPDMS油及び硬化剤を混合する。徹底的に両方の化合物を混ぜて。 </li><li>金型ベアリングマイクロチャネル上でミックスをキャスト。全体の高さは0.5〜1センチメートルの間でなければなりません。 </li><li> 1時間の間に真空ジャーベルで気泡を除去。 </li><li> 65℃で2時間オーブンで後者を配置することによって、金型で硬化PDMS </li><li> PDMSは、室温であると、外科用ブレードを用いて構造の周りに大きな部分をカットして、金型（ 図1A）からそれをはがす。 </li><li>細胞は（通常は2ミリメートル）を注入し、PDMSのサイズを変更されるドリル穴移行が評価されるであろうガラスボトムディッシュ（ 図1B）のサイズに合わせて、外科ブレード。先に進む前に、レンズクリーニングペーパーを使って皿から残っている埃を取り除きます。これは、ステップ1.10に記載のガラスへのPDMSの結合を促進する。 </li><li>構造側に粘着テープを貼り付けし、剥離することによりチャネルを含むサイズ変更PDMSチップを清掃してください。 </li><li>粉塵及びPDMS小粒子を除去するために70％エタノール中でPDMS片を30秒間超音波処理する。きれいな空気を吹き付けて、その後すぐにそれらを乾燥させます。 </li><li> 300トルで30秒の間にプラズマ処理空気（または酸素）によって、PDMS（上向き構造体）と培養皿をアクティブにします。 </li><li>恒久的に基板に、PDMSを固執する接触の両方で活性化された表面に置きます。必要に応じて、ポリマーおよびディッシュ（ 図1C）のガラスとの間の接触を強制的にPDMSの上に少し押して、金属鉗子を使用しています。 </li><li> 65°CのFOのオーブンでチップをインキュベート結合を強化するため、R 1時間。 </li></ol> 2。マイクロチャネルのコーティング<ol><li> 1分間300トルで空気プラズマにより全体構成をアクティブにします。これは次のステップのチャンネルへの液体の侵入を推進していきます。 </li><li>水中で10μg/ mlので急速にフィブロネクチンとチップのエントリ穴を埋める。例えば、PEGのような他の基質は、チャネル壁への細胞接着を修飾するために使用することができる。液体は全体の構造全体に広がることを注意してください。これは簡単に、通常の光の下または通常の明視野顕微鏡を用いて目で確認することができます。 NOTE：拡散が困難になっている、非常に小さい構造では、チャネル内の液体の侵入を少なくとも15分の間に真空ジャーベル構造を配置することによって強制することができる。コーティング蛍光基質の効率を検証する（ 図2）を用いることができる。 </li><li>フィブロネクチンの吸着または任意の他のコーティングにsubstを可能にするために室温で1時間インキュベートチャネルの壁に対する割合。 </li><li>構造が非結合基材を除去するために、PBSで3回洗浄します。 </li><li>洗浄後、議定書3に進むか、後で使用する（24時間の最大値）、4℃でチップを格納します。 </li></ol> 3。セル負荷<ol><li>プレートからPBSを除去して、細胞培地でマイクロシステムを埋める。媒体とのチャネルを飽和させるために1時間インキュベートしてみましょう。 注：分子阻害剤のような薬物を含む実験のためには、右の濃度の薬剤を含む培地でチャンネルをプレインキュベートすることをお勧めします。さらに、いくつかの薬物は、有効濃度を低下させるPDMS構造体に可溶化する傾向がある。一部のソリューションは、この問題11-12に対抗するために提案されている。 </li><li>浮遊DCを削除し、培養液でフラッシュすることにより、半接着細胞を回収する。血球計数器を用いて細胞を数える。 注：核イメージングでは、ヘキスト33342染色はインで事前に達成することができ完全培地中の200 ng / mlの時に染料と30分の間に2×10 6の細胞をcubating。プロトコルを続行する前に色素の過剰を取り除くために遠心分離により二回洗浄します。 </li><li> 5分（時間および速度は細胞の種類に応じて異なっていてもよい）中に300×gで細胞を遠心し、培地を捨てる。 20×10 6細胞/ mlの濃度に達するようにペレットを希釈する。 </li><li>プレートから培地の過剰を削除し、マイクロピペットを用いて、PDMS構造のエントリの穴を空にします。各エントリの穴に1×10 5個の細胞の量に到達するために細胞溶液5μlのエントリを入力します。 NOTE：高細胞密度は、チャネルによる細胞の接触に有利に働くことが要求される。低細胞密度は、チャネルと、実験の失敗の内側の細胞の少数をもたらし得る。 </li><li>インキュベーター内で37℃で30分間、マイクロチップをインキュベートする。実験皿に完全培地の2ミリリットルを追加します。 注：全体のPDMS構造は、bになりませんeは実験中の細胞の乾燥を避けるために覆わ。 2mlの十分でない場合、完全にPDMS構造体を覆うように、より多くの培地を加える。 </li></ol> 4。イメージング<ol><li>顕微鏡下のプレートを配置する前にレンズクリーニングティッシュと料理の外部底面を清掃してください。 </li><li>多数の細胞内移行を分析するには、CO 2と温度を装備ビデオ顕微鏡で10倍と広い視野照明（ 図3）を使用します。細胞追跡を容易にするために、ヘキスト染色およびUV光は（ステップ3.2参照）を使用することができる。移行は、移行​​中に細胞構造の高解像度を得るために、共焦点顕微鏡を用いて分析することができる。 </li><li> 10Xのタイムラプス顕微鏡検査のために、期待されるセル速度（5ミクロン/分の速度で移行した樹状細胞のため、通常2分）に応じた時間周波数を選択します。 注：ここに記載されているプロトコルは、nを行いますオトは、細胞遊走パラメータの分析を含む。いくつかのポイントは、時間経過の映画から情報を抽出するためにどのように進めるかについて、読者に助言するために議論に記載されています。 </li></ol>

<ol>
	<li>Lauffenburger, D. A., Horwitz, A. F. <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:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84 (3), 359-369 (1996).</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>Schor, S. L., Allen, T. D., Harrison, C. 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Immunol. 30, 1-22 (2012).</li><li>Faure-Andr&#233;, G., 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=Regulation+of+dendritic+cell+migration+by+CD74,+the+MHC+class+II-associated+invariant+chain.">Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain.</a> Science. 322 (5908), 1705-1710 (2008).</li><li>Renkawitz, J., 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=Adaptive+force+transmission+in+amoeboid+cell+migration.">Adaptive force transmission in amoeboid cell migration.</a> Nat. 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著者らは、開示することは何もありません。

ここでは、単一の実験で多数の細胞の遊走特性を研究するための方法として、マイクロチャネルからなる装置を記載している。この実験系は、内因性の遊走細胞によって組織に見閉じ込められた環境制約を模倣。しかし、単一の次元に移行を強制することにより、自動細胞追跡とmeasurablesの抽出（ 図5）を容易にします。我々はまた、我々のデバイスは、蛍光顕微鏡と互換性があ?...

マイグレーションは、開発、免疫応答および組織再生を含む多細胞生物における多くの生理学的プロセスに重要である複雑な細胞機能である。また、このような腫瘍の浸潤および転移などの特定の病的状態は、細胞運動性1に依存している。これらの理由から、細胞遊走、基本および翻訳の両方の研究との関連で研究の主要な分野となっている。 インビボでは 、ほとんどの組織が ​​豊富な細胞外マトリックスおよび高細胞密度によって特徴付けられる。細胞移動は、従って、生理的条件下で、複雑な閉じ込められた環境で発生する。古典的には、最も可能性の高い歴史的な理由や技術的な限界のために、細胞移動は、監禁などの組織に見られる環境プロパティの多くを再現していないフラットな2D系で研究されている。また、2Dでの運動性のために必須である細胞接着などの要因は、最近necessaではないことを示したされていますRILY 2Dにし、他の環境での細胞運動を支配する機構は2異なっていることを示唆し、 生体内でまたはゲルの内部移行に必要。いくつかのシステムは、細胞外マトリックス組成物3の特性をrecapitulating目指す組織の複雑な性質、最も有名なものコラーゲンゲルを模倣するために開発されている。ここでは、限られた環境下で1次元での研究細胞移動を可能にする簡単な補完的な方法として、マイクロチャネルを提案する。 このシステムでは細胞は、それらが自然に入るその中にマイクロチャネルに沿って移動する。遊走細胞はその後、おそらくそれらの極性を強化管状ジオメトリを採用し、チャンネルの形状を取得する。チャネル内のセルの直線運動は、自動細胞追跡実験からの定量パラメータの抽出を可能にする。技術的な観点から、このシステムは、簡単で柔軟性があります。 coatinチャネル壁のgを操作することができ、チャネルのサイズおよび形状を適合させることができ、多数のセルは、単一の実験で分析することができる。このシステムはまた、スケールアップすることができる細胞の運動性に関与する分子の中距離スクリーン解析を実行する。ここで説明するプロトコルは、細胞モデルとして樹状細胞（DC）を用いて標準化されている。彼らは4。 インビトロ 、DCは自然発生的に限られた環境の中を移動することが示されている特異的な免疫応答の開始と維持に参加し、したがって、マイクロチャネル5,6に細胞運動を研究するための優れたモデルであるように、これらの細胞は、免疫システムの鍵となる。重要なことに、このシステムは、Tリンパ球、好中球、または腫瘍細胞を7-9などの他の運動性の細胞型の遊走を分析するために拡張することができる。

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:273px;">PolyDimethylSiloxane (PDMS)</td>
			<td style="width:131px;">GE Silicones</td>
			<td style="width:139px;">RTV615</td>
			<td style="width:405px;">Package of 90% silicone base and 10% curing agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Core sample cutter</td>
			<td>Ted Pella Int.</td>
			<td>Harris Uni-Core&#160;</td>
			<td>Diameter 2,5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass-bottom dish</td>
			<td>WPI</td>
			<td>Fluorodish FD 35-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic cleaner</td>
			<td>Branson Ultrasonics</td>
			<td>Branson 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC 32 G</td>
			<td>For small samples (35 dishes). A bigger version is also available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PolyLysine grafted PEG (Pll-g-PEG)</td>
			<td>Susos</td>
			<td>PLL(20)-g[3.5]-PEG(5)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y27632</td>
			<td>TOCRIS</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of cell migration in microfabricated channels

ここで説明する方法は、1次元での閉じ込め下で細胞遊走の研究を可能にする。これは、物理的な制約によって細胞に偏光された表現型を与える微細加工されたチャネルの使用に基づいている。前後に移動：内部のチャンネルと、細胞は2つの可能性を持っている。指向性が制限され、この簡略化は、細胞遊走の自動追跡および細胞移動を説明するための定量的なパラメータの抽出を容易にする。これらのパラメータは、運動中の細胞の速度、方向の変化、一時停止が含まれる。マイクロチャネルはまた、蛍光マーカーの使用と互換性があり、したがって、高解像度での細胞移動の際に細胞内小器官及び構造の局在化を研究するのに適している。最後に、チャネルの表面は、チャネルまたは走触性の研究の粘着特性の制御を可能にする、異なる基質で官能化することができる。要約すると、システム時間記載やがては独立した実験の標準化と再現性を容易にする、幾何学的形状と環境の生化学的性質の両方が制御された条件で大細胞数の移動を分析することを意図している。

各実験において、PDMSの表面は。前と（ステップ2.4）で洗浄した後、図2に示す蛍光分子、PLL-gの-PEGでコーティングされたチャネルを図調査の関心に適合分子でコーティングされる。このような実験は、チャンネル内のコーティングの均一性を制御することができる。 セルの負荷後に、ビデオ顕微鏡は、細胞移動に追従して行うことができる。 <str...

Watch this Scientific Journal Video about Study of Cell Migration in Microfabricated Channels at JoVE.com

Study of Cell Migration in Microfabricated Channels

一次元閉じ込められた微小環境における細胞の自発的な遊走を研究するための定量的方法が記載されている。この方法は、微細加工されたチャネルを利用し、単一の実験では、異なる条件下で多数の細胞の遊走を研​​究するために使用することができる。

微細加工チャネルにおける細胞移動の研究

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

study-of-cell-migration-in-microfabricated-channels

Research

JoVE Journal

Bioengineering

11.8K ビュー.  Institut Curie. 一次元閉じ込められた微小環境における細胞の自発的な遊走を研究するための定量的方法が記載されている。この方法は、微細加工されたチャネルを利用し、単一の実験では、異なる条件下で多数の細胞の遊走を研​​究するために使用することができる。 

ビデオ: Study of Cell Migration in Microfabricated Channels

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

機械的にダイナミックな細胞培養のための微細加工プラットフォーム

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

生物工学

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

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 ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

蛍光顕微鏡を用いたイメージング細胞遊走用接着剤及び可溶性グラデーションを作成する

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

同時にデュアルカメラエミッション分割システムを使用して、2つの発光チャンネルにリアルタイム画像のキャプチャ：アプリケーションは、接着性を細胞する

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

における損傷応答のライブイメージングや研究のためのマイクロ流体チップを使用<em&gt;ショウジョウバエ</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

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 ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心円状のゲルシステムは、3D細胞移動に対するマトリックスの微小環境の生物物理役割を研究する

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

骨様テンプレートでのマイクロチャネルによって独特の毛細管現象は大骨欠損の回復のために細胞の動員を強化することができます

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製<em&gt;インビボ</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Methods Article

微細加工チャネルにおける細胞移動の研究