著者らは、彼らのサポートのためにメンフィス大学の技術のフェデックス研究所に感謝そのたいと思います。著者らは、メンフィス（デビッド·バトラー、ジャッキー·カーター、ドミニククリーブランド、ヤコブシェーファー）の大学で機械工学部門のシニア·デザイン·プロジェクトグループの学生を認めるしたい、モータ制御のための大学メンフィスの技術工学部門からのダニエル·コーン細胞培養における彼らの助けのため、博士ビンテンさんとCharlean Luellen。この作品は、K01 HL120912（ER）とR01 HL123540（CMW）によってサポートされていました。

1。細胞培養培地の保持のためのウェル壁を有する膜の構築（最終製品については、図1Dを参照してください） <ol><li>コラーゲンIでコーティングされたポリジメチルシロキサン（PDMS）のシートを使用して、外科用メスまたはダイを可撓性膜の輪郭をカットします。 </li><li>ストレージのための60ミリメートルペトリ皿に各膜を配置します。 </li><li>壁の作成： <ol><li> B（硬化剤）をエラストマーにエラストマーAの1の重量比：10にPDMSを混ぜます。 </li><li> 50mlチューブに完全に混合PDMSの5ミリリットルを注ぎます。 </li><li>ハイブリダイゼーションオーブンに水平に未硬化のPDMSを用いて50 mlチューブを置きます。 </li><li>硬化時間の間に8回転で被覆するために、チューブの内壁をローター関数を使用します。 RTで、PDMSは、完全に2日間で硬化させることになります。 </li><li>滅菌細胞培養フード内管からPDMSを削除します。 </li><li>新しいカミソリの刃を使用して、セクションの高さ4ミリメートルにPDMSのシリンダーを分割。 </li><li>膜WAとなるセクションを配置壁が折り目を付けになることを可能にすることのないペトリ皿容器にLLS、。 </li></ol></li><li>ペトリ皿に2を維持しながら、一つの膜の上にPDMSの一方の壁を選択し、中央。 </li><li>接着剤として使用する壁の外側周囲に：（1比10）静かに未硬化PDMSを配置します。それは、液体を保持することがあるため、壁と膜との間のギャップの形成を防止します。 </li><li>各ペトリ皿完成膜を含有し、硬化させる24時間70℃のオーブンでカバーを置きます。 </li></ol>キャリブレーションのためのクランプ変位または放射状の成長（図2）とモータ回転2.相関<ol><li>モータを制御するソフトウェアを起動します。 </li><li>モーターソフトウェアの手動設定を使用してデバイスのクランプを移動さ。 </li><li>距離を測定し、クランプの最小値と最大変位の両方でクランプの対向間のモータカウント位置を記録。 </li><li> Dの変化率（％）を計算しますモーター·カウント（〜0-75kカウント）の関数としてのクランプ間istance。これは、膜上で達成される最大の潜在的なひずみを示します。 </li></ol>マウス肺上皮細胞株のストレッチの3.アプリケーション（MLE12） <ol><li>ウェルあたり可撓性膜上の250万細胞（ステップ1）でのシードMLE12細胞は、2日以内にコンフルエントします。播種密度は、異なる細胞タイプについて変化し得ます。 </li><li>機械式アクチュエータが完全にリラックスした位置にあることを確認します。 <ol><li>細胞培養培地を除去します。 </li><li> 6クランプのそれぞれに2つの穴を開け、1.5ミリメートルに生検パンチを使用して膜のタブ（ 図1Dを参照してください）。穴の径方向の配置は、最初にプリテンション膜経験の量を決定します。何の予備張力を希望されない場合、膜のタブで20.5ミリメートルの半径で穴を開ける。 </li><li>パンチ穴がクランプ内ピンと並んでストレッチャー上の膜の位置を調整します。代わりにトップクランプを配置します。ネジの側面を交互に一つずつを締めます。 </li><li> 1ミリリットルの細胞培養培地を追加します。 </li></ol></li><li>光路を有する膜の中央を中心に顕微鏡ステージ上には置か。 </li><li> （可能な場合）ステージにデバイスを固定するテープや磁石を使用してください。いったん固定、顕微鏡のステージコントローラと機械装置の面内（膜に平行）および垂直（Z方向）を制御します。 </li><li>手動でモータ回転20の位置や速度を制御することにより、製造業者によって提供されるソフトウェアでストレッチを適用します。 </li></ol>ミトコンドリア活性酸素種の4測定（ROS） <ol><li>細胞がコンフルエントになったら、直接細胞にミトコンドリアのスーパーオキシドインジケータ（5μM最終濃度）でRT DMEM 1〜2mlのを追加します。 </li><li> 37℃で10分間インキュベートします。 </li><li>洗浄細胞を穏やかに緩衝液で3回の水浴中で加温し37°C。 </li><li>すぐに直立共焦点顕微鏡下で既に代わりにストレッチャ（ステップ2.2）に柔軟な膜を配置します。 </li><li>フェノールレッドを含まない培地およびHEPES 25mMので1mlのDMEMを追加します。 </li><li> 580分の510 nmの励起/発光フィルターを設定します。 </li><li>画像の複数のフィールドは、15分ごとには、ミトコンドリアのスーパーオキシド産生の所望の時間経過を作成します。 </li><li>撮影された画像から、記録蛍光強度は、蛍光強度を定量化することが可能なソフトウェアを使用して、各時間間隔でヒストグラム。 </li></ol>ストレッチや原子間力顕微鏡（AFM）の5.アプリケーション注：これらの手順は、特定のAFMと光学顕微鏡の組み合わせ（ 図4および物質一覧）のために提供されます。 <ol><li>実験のAFMを準備します。 <ol><li> z方向の最大位置にAFMヘッドの高さを増やします。 </li><li> AFMの足への増量を入れてサンプルAFMカンチレバーコンタクトれるプレーンを持ち上げます。試料とAFMヘッドは機械装置の高さに対応するために持ち上げられる必要があります。 </li></ol></li><li> （利用可能な場合）、光学顕微鏡を準備します。 <ol><li> AFMスキャナプレートを取り外します。所望の目的を削除します。 </li><li>客観的にスペーサーを追加します。スペーサの高さは、客観的かつ具体的なAFMのセットアップに依存しますが、光学イメージングが所望される場合、観察面がストレッチャーとアダプター高さに等しい量だけz方向にシフト（ 図になりますので、それが必要です図5（a））。 AFMは、通常、デバイス上記光路から低倍率のイメージングを提供することに注意してください。 </li><li>バックの位置で所望の目的をマウントします。 AFMに戻すにスキャナを配置します。 </li><li> AFMソフトウェアを起動します。蛍光測定のための光源を含むすべての必要な光源を起動します。 </li><li>その片持ち梁にチップをマウントします目的の測定に適しています。生細胞の弾性率を測定するとき200 PN / nm以下剛性が好ましいです。 </li><li>レーザーの位置を合わせ、デバイスに搭載されたガラスカバースリップ上で製造者の指示に従ってカンチレバー剛性を調整します。 </li></ol></li><li>ステップ1のものが、以下のように変更して膜を準備します。 <ol><li>すぐにストレッチデバイス上に膜を取り付ける前に、高さ約1mmの壁をカット。これは、膜の壁とロードセルとの間に経験する干渉を防止します。 </li><li> 3.2.1-3.2.3に記載されているように機械装置の膜をマウントします。 </li><li>流出の場合にはAFMスキャナへの損傷を防ぐために、細胞培養培地を取り除き。 </li><li>カップルアダプタ（ 図5A）を持つデバイス。スキャナーのアダプタと機械装置を配置します。 </li><li>希望の引張ひずみレベルに膜を伸ばします。 </li></ol></li><li&gt;延伸した細胞のナノインデンテーション。 <ol><li>による流出にAFMスキャナや顕微鏡の損傷を避けるために、細胞上のメディアの限定（&lt;0.5 ml）のボリュームを追加します。 </li><li>膜と片持ち梁に係合します。 </li><li>関心領域をスキャンするために、特定のAFM装置のプロトコルに従ってください。 </li></ol></li></ol>

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著者は、彼らが競合する金融利害関係がないことを宣言します。

メカニカルストレッチ中に生細胞イメージングのためのユニークなデバイスを開発しました。このデバイスは、肺上皮細胞のメカノを研究するためのプロトコルで使用されました。予備研究では、単一の保持されたストレッチは、気管支上皮細胞におけるミトコンドリアのスーパーオキシド産生を刺激することが見出されました。また、機械的な歪みのレベルの増加は、肺胞上皮細胞の単層?...

細胞は、多くの組織における機械的負荷にさらされ、この機械的刺激は、遺伝子発現、増殖因子の放出、サイトカイン、または細胞外マトリックスと細胞骨格のリモデリング1-4のパターンの変化を促進することが示されています。このような機械的刺激から形質細胞内シグナルは、メカノ5-7のプロセスを経て起こります。呼吸器系では、メカノの一つの結果は、活性酸素種（ROS）の環状の引張歪みの存在下での肺上皮細胞における8,9およびプロ炎症性サイトカイン10の増加です。強力な証拠も過度の引張歪みがセル11-14の生化学的応答に加えて、肺胞上皮の損傷を導くにつながることを示唆しています。ここでの焦点は、機械的変形に肺細胞の応答に主であるが、メカノによって誘発される経路は、BASにおいて重要な役割を果たし血管緊張15の調節および成長板16の開発など、人体の多くの組織のIC機能。 メカノへの関心の高まりは、培養細胞や組織への生理学的に関連する機械的負荷の適用のための多数のデバイスの開発をもたらしました。具体的には、組織が ​​経験する機械的負荷の一般的な形態である引っ張り歪を適用するデバイスは、人気11,17-19あります。しかし、利用可能なデバイスの多くは、いずれかの組織工学アプリケーションのためのバイオリアクターとして設計やストレッチでリアルタイムイメージングに資するものではありません。このように、メカノの経路の調査を容易にするために、張力の細胞及び組織を視覚化することができるツールおよび方法を開発する必要があります。 ここで、面内の機械的延伸装置が設計され、プロトコルは、Mを適用するために開発されましたultiple組織への株の形態および細胞リアルタイム（ 図1A-D）における生化学的および機械的応答のイメージングを可能にします。デバイスは、可撓性膜をつかみ、約20％（ 図1B）に面内、ラジアル膨満アップを適用するために円周方向に配置された6つの等間隔のクランプを使用しています。モータ（ 図1C）がインキュベータの外 ​​部に配置され、モータ供給業者によって提供される独自のソフトウェアによって制御される作動装置は、長期間の細胞培養インキュベーターに置くことができます。モータは、緊張と弛緩中に均一6ストレッ​​チャクランプを駆動する、内部カムを回転させるリニアドライバに接続されています。 機械装置に加えて、カスタマイズされた柔軟な膜は、機械的なシステムで使用される市販の細胞培養準備膜から作成されました。およその直径そして円形壁（28ミリメートル）を作製し、細胞にのみ十分に記載ひずみプロフィールのこの領域中で培養することができるように、可撓性膜上に付着させました。作動装置内のこれらの膜の配置は、可撓性膜の中心部に均一で等方性の歪みを提供するかどうかを決定するために、有限要素解析は、市販のソフトウェア（ 図1E-F）を用いて行きました。フレキシブル膜は、対称境界条件でモデル化し、メッシュのすべての四角形要素を利用しました。 図1Fに示す最大主ひずみの等高線図に見られる同心リングは、歪みの等方性の分布を示しています。 膜によって受ける歪みがローディング（ 図2）を介してマーキングの画像を記録することによって測定した。 図2Dは ​​、半径方向及び軸方向に測定された平均膜株がほぼ直線であったことを示します適用モータに対して20％の最大線形歪みにカウントアップします。バック休止位置へ後退時に測定されたものと比較して膨張時に測定された歪みのレベルの間に有意差はなかったです。次に、カスタム可撓性膜上で培養したヒト気管支上皮細胞（16HBE）及びそれらの核の変位を測定しました。全細胞の変位は、デジタルマイクロスコープを用いて記録位相コントラスト画像を用いて測定したのに対し16HBE細胞の蛍光標識（DAPI）核は、共焦点顕微鏡下で20X対物レンズを用いて画像化しました。 図3に見られるように20％の線形歪み〜まで、核の変位によって測定ひずみは、膜上のマーキングの変位によって測定されたものと同様でした。これは、膜に加わる歪みを接着細胞に伝達されたことを確認します。従来の顕微鏡のカスタムデバイスの使用を記述プロトコルおよび原子間力microscopeは、以下のステップで提供されます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SmartMotor NEMA 34: 3400 Series</td>
			<td>MOOG Animatics</td>
			<td>SM3416D</td>
			<td>Integrated motor, controller, amplifier, encoder and communications bus</td>
		</tr>
		<tr>
			<td>Flexcell Membrane (Collagen I coated)</td>
			<td>Flexcell International Corp</td>
			<td>SM2-1010C</td>
			<td>3.5x5.25x0.020&#34;</td>
		</tr>
		<tr>
			<td>Sylgard 184&#160;</td>
			<td>Dow Corning Corporation</td>
			<td></td>
			<td>10:1</td>
		</tr>
		<tr>
			<td>Hoechst 33342&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H1399</td>
			<td>DAPI stain</td>
		</tr>
		<tr>
			<td>MitoSOX</td>
			<td>Sigma-Aldrich</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiron</td>
			<td>Sigma-Aldrich</td>
			<td>D7389&#160;</td>
			<td>mitochondrial superoxide label</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td></td>
			<td>superoxide inhibitor</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-19</td>
			<td>Any centriguge tube can be used to create an area for imaging.</td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Bellco Glass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MLE12 Cells</td>
			<td>ATCC</td>
			<td>CRL-2110</td>
			<td>Mouse Lung Epithelial Cells&#160;</td>
		</tr>
		<tr>
			<td>16HBE cells</td>
			<td>ATCC</td>
			<td>CRL-2741</td>
			<td>Human Bronchial Epithelial Cells</td>
		</tr>
		<tr>
			<td>AFM Indentation Experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cantilever Beams for Nano-indentation</td>
			<td>Budget Sensors</td>
			<td>Si-Ni30</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM&#160;</td>
			<td>Asylum Research</td>
			<td>MFP3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td>Inverted microscope with 20X and 40X objectives.</td>
		</tr>
		<tr>
			<td>AFM Leg Extenders</td>
			<td>Asylum Research</td>
			<td>Not available</td>
			<td>AFM microscope</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finite Element Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABAQUS</td>
			<td>Simulia</td>
			<td>6.12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Life Technologies</td>
			<td>EVOS XL Core</td>
			<td>Initially a self standing company, now owned by Life Technologies.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 710</td>
			<td>2-photon upright microscope</td>
		</tr>
	</tbody>
</table>

live cell imaging during mechanical stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

活性酸素種と変形 以前の研究は、環状のストレッチ21に応答して、気道及び肺胞上皮細胞における活性酸素種（ROS）の増加を示しました。活性酸素種は、分子及び脂質、タンパク質、多糖、および核酸22-24に対して高い反応性を有する分子酸素由来のフリーラジカルを含みます。 ROSは、一般的な細胞内のイオンチャネルの機能を調節するための信?...

Watch this Scientific Journal Video about Live Cell Imaging during Mechanical Stretch at JoVE.com

Live Cell Imaging during Mechanical Stretch

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

メカニカルストレッチ中のライブセルイメージング

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

live-cell-imaging-during-mechanical-stretch

Research

JoVE Journal

Bioengineering

10.3K ビュー.  University of Tennessee Health Science Center. A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy. 

ビデオ: Live Cell Imaging during Mechanical Stretch

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

軸索のストレッチの成長：神経成長のメカノトランスダ

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

生物工学

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

三次元マトリックスの蛍光タグ付きタンパク質を発現する遊走細胞の生細胞イメージング

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

容器内ステントの展開中の壁の力学を監視する

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

ハイスループットマイクロカプセル化シングルセルと複数セル

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

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 ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

マイクロ流体技術は、Cell変形能の探査

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

商業顕微鏡でのライブ細胞膜上の分子拡散法への高速蛍光イメージングから

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

赤血球凝集の動的な調査のための制御されたマイクロ流体環境

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

病の診断のためのポータブルイメージングおよび分析と相まって磁気浮上

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Isokinetic Robotic Device to Improve Test-Retest and Inter-Rater Reliability for Stretch Reflex Measurements in Stroke Patients with Spasticity

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has been shown to be limited in inter-rater reliability. One factor in this poor inter-rater reliability is the variability of passive motion while measuring the angle of catch (AoC) measurements. Therefore, an isokinetic device has been proposed to standardize the manual joint motion; however, the benefits of isokinetic motion for AoC measurements has not been tested in a standardized manner. This protocol investigates whether isokinetic motion itself can improve inter-rater reliability for AoC measurements. For this purpose, a robotic isokinetic device was developed that is combined with surface electromyography (EMG). Two conditions, manual and isokinetic motions, are compared with the standardized method to measure the angle and subjective feeling of catch. It is shown that in 17 stroke patients with mild elbow flexor spasticity, isokinetic motion improved the intraclass correlation coefficient (ICC) for inter-rater reliability of AoC measurements to 0.890 [95% confidence interval (CI): 0.685&#8211;0.961] by the EMG criteria, and 0.931 (95% CI: 0.791&#8211;0.978) by the torque criteria, from 0.788 (95% CI: 0.493&#8211;0.920) by manual motion. In conclusion, isokinetic motion itself can improve inter-rater reliability of AoC measurements in stroke patients with mild spasticity. Given that this system may provide greater standardized angle measurements and catch of feeling, it may be a good option for the evaluation of spasticity in a clinical setting.

Using a robotic isokinetic device with electromyography (EMG) measurements, this protocol illustrates that isokinetic motion itself can improve inter-rater reliability for the angle of catch measurements in stroke patients with mild elbow flexor spasticity.

痙攣性脳卒中患者におけるストレッチ反射測定のテスト再テストと評価間信頼性を向上させるアイソキネティックロボット装置

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has ...