NIHの助成金HD052670でサポートされています。

1。 ECMタンパク質とプレートのコーティング<ol><li>無菌条件下で、プレート当たり冷滅菌1X PBS 12 mlでラミニン120μgを混合します。 </li><li> Bioflex未処理の板を取り、各ウェルに溶液の2ミリリットルを追加します（ラミニンの最終濃度は2μg/ cm 2である）。また、他のECMタンパク質は、コラーゲン-1 [10μgの/ cm 2]と、フィブロネクチン[を5μg/ cm 2]と、ビトロネクチン[0.5μgの個/ cm 2]またはエラスチン[10μgの個/ cm 2]のように、使用することができます。コー​​ティング技術は、それがラミニンコーティングのために説明されているものに似ています。井戸は完全に溶液で覆われていることを確認してください。 </li><li>プラスチック製のプレートをラップし、ウェルの底に吸着ラミニンを可能にするために一晩、平らな面に4℃で置く。まだ良いECMの機能を維持しながら、これらの条件において、プレートは少なくとも1週間保存することができます。 </li><li>翌日、無菌条件下で、1X PBSでウェルを3回洗浄します。 </li><l私は&gt;を各ウェルに1×PBSで1％のBSAを1mlを加え、37℃でインキュベート膜上の非特異的結合部位をブロックするために1hの°C。の</li><li>非吸着タンパク質を除去するために1X PBSでプレートを3回すすいでください。 </li><li>細胞は、ステップ2.14から隔離されるまで37℃DMEM中℃でプレートを保持します。 </li></ol> 2。胎児の齧歯類の肺II型上皮細胞の分離分離前の日は、オートクレーブと準備ができて、異なるサイズのナイロンメッシュを使用して、画面のコップを持っています。 <ol><li>妊娠のE17-19の上でマウス/ラット時限妊婦から胎児肺を取得します。 DMEM培地を含む50 mlのコニカルチューブに組織を移し、氷の上に置きます。 </li><li> 50 mlコニカルチューブに消化バッファーを作成します。（10ミリリットル）。このボリュームには、マウス/ラットから約20胎児からの肺組織が使用されます。 8.5ミリリットルDMEM 0.5ミリリットル500MM pH7.4のHEPES 5 mgのコラゲナーゼ1 5 mgのコラゲナーゼ1A 1ミリリットル鶏血清、不活化熱</li><li>すべてのコンポーネントが、無菌フード内0.2ミクロンのシリンジフィルターを通して溶解し、フィルターされるまでよく混ぜる。 37℃の水浴中で消化緩衝液を含むコニカルチューブ℃を置く</li><li>ステップ2.1からコニカルチューブからDMEM培地を除去し、滅菌シャーレに組織を転送します。 </li><li> 1mm以上の組織片は、以下のように、ステップ2.3から予め温めておいた消化緩衝液を含むコニカルチューブに組織を転送するために滅菌済みのハサミかカミソリの刃でも肺をミンチ。 </li><li>上下にピペッティングによって組織を消化する。 10ミリリットルピペットで100倍 5ミリリットルピペットで100倍 パスツールピペットで100倍</li><li>室温で5分間1300 rpmでホモジネートを遠心分離します。 </li><li>慎重に吸引して上清を除去し、ペレットをDMEMを15ml加え、20％FBSで細胞を含む再懸濁する。 </li><li> 100画面のコップを取り出し、 - 、30 - 、15ミクロンのナイロンメッシュと場所番目滅菌した150mlのビーカーにEM。 </li><li>ステップ2.8から細胞ホモジネートを追加し、順次フィルタ100を介して - 、30 - 、15ミクロンのメッシュスクリーンカップ。 </li><li>非上皮細胞のろ過を容易にするために、DMEM +10％FBSで数回洗浄した後、15ミクロンメッシュ - 凝集30から非濾過し、細胞を収集します。 </li><li>主に線維芽細胞が含まれている15ミクロンメッシュからろ液を捨てる。 </li><li> 30分（フラスコ当たりの細胞懸濁液のapproximatelly10 ml）を75-cm 2のフラスコにステップ2.11から細胞をインキュベートすることにより、さらにII型細胞を精製する。 </li><li>ペレット細胞に室温で5分間1300 rpmで遠心し上清を収集します。 </li><li>慎重に吸引して上清を除去し、ペレットを無血清DMEM中で細胞を含む再懸濁する。再懸濁の量は、処理された組織の量に依存します。およそ、私たちプレートは、細胞と同等の阿智のために（2 ml）を各ウェルに1つの胎児から得られた次の日にコンフルエントに百分の60から70の間で前夜。 </li><li>ラミニンまたはフィブロネクチンでプレコートBioflex 6ウェルプレート上にプレートの細胞懸濁液。 </li></ol> 3。胎児のげっ歯類の肺線維芽細胞の分離<ol><li> 2.11までのII型細胞の単離のために上記と同じ手順に従ってください。 </li><li>線維芽細胞が付着できるようにするために30〜60分間37℃で75-cm 2の上に、プレートを、15ミクロンメッシュ（おおよその体積は10〜15 mlで前洗浄せず）からのろ液を取る。 </li><li> supernantantを吸引除去し、無血清DMEMと交換して、一晩インキュベートする。 </li><li>次の日、0.4mMのEDTAおよびプレートそれらのフィブロネクチンでプレコートBioflexプレート上で0.25％（wt / vol）のトリプシンで細胞を収穫します。 </li></ol> 4。肺細胞に機械的刺激を提供するための実験系<ol><li>種子無血清DMEMでBioflex培養プレート上で細胞（約50％コンフルエント）（2ミリリットル/ウェル）し、それらを添付してSPREう実験前少なくとも6時間の広告。一般的に私たちの研究室では、機械的歪みを適用する前に、約24時間の培養中の細胞を維持しています。特定のセルの数は実験に必要な場合は、分離した後、II型細胞は無血清DMEM 75-cm 2のフラスコと次の日に保持され、細胞をトリプシン処理し、計数し、その後に播種されています。 </li><li>翌日、メディアは新鮮な無血清DMEM（2ミリリットル/ウェル）とbioflexプレートに置き換えられますがFlexcell FX-5000ひずみユニットに搭載されている。単分子膜は、実験の開始前に超えない80％コンフルエントでなければなりません。 </li><li>等二軸ひずみが膜に適用されます。株のレジメンは（説明を参照してください）in vivoでの機械的な力のシミュレーションによって異なります。 </li><li>並列で培養した無延伸膜上に培養した細胞を対照として使用されています。 </li><li>実験の終わりに、単分子膜は、遺伝子発現の変更（例えば、界面活性プロテインCのために）分析するために処理することができますリアルタイム-PCR、ウエスタンブロット法によるタンパク質の豊富さなどによっても、組織化単分子膜は、免疫細胞化学実験のために固定することができます。この手法では、固定した後、シラスティック膜は板から切り出しており、抗体を用いた透過性とインキュベーションの前に取り付け剤としての水の10〜20μlを用いてスライドガラス上にマウントされている。上清はまた、成長因子、サイトカインなどの存在を調査するために使用することができます</li></ol> 5。代表的な結果 E18胎児マウスのII型細胞の図1と図2には、show代表的な位相コントラストの写真は、この原稿で説明する手法を用いて単離された。 図3は、その機械的ひずみがマーカーとして界面活性剤、タンパク質-Cを使って胎児のII型上皮細胞の分化を誘導を示しています。 <img src="/files/ftp_upload/3543/3543fig1.jpg" /> 図1。担当者resentative位相コントラストの写真は、右胎児のII型細胞の凝集外観を示す分離後に撮影。 <img src="/files/ftp_upload/3543/3543fig2.jpg" /> 図2：E18胎児のII型上皮細胞は、ここで説明したように単離され、ラミニンでコーティングされたbioflexプレート上に播種した。無延伸細胞はパラホルムアルデヒドで固定した後の写真は、次の日に撮影された。細胞の純度は90であることが決定された±5％をSP-Cの上皮細胞の形態や免疫染色の顕微鏡分析によって。 <img src="/files/ftp_upload/3543/3543fig3.jpg" /> 図3。胎児II型上皮細胞を16時間40サイクル/分で5％、環状の歪みにさらされた。そのひずみを示す界面活性プロテインC（SP-C）mRNAの発現）ノーザンブロットは、異なるECM基板を用いたII型細胞の分化を誘導する。 + / - 記号は、sへの暴露を表すかどうかそれぞれ電車、。データは+ /を意味するとして表示されます- SEM、n = 3で、* P &lt;0.05 B）蛍光免疫細胞化学画像は機械的ひずみにさらされるかどうか（コントロール）胎児のII型細胞でSP-C蛋白質レベル（緑）を実証する。核はDAPI（青）で対比染色した。バーは、そのメカニカルストレッチを示す3つの実験から10μmである。C）ウェスタンブロットの結果は、SP-C蛋白質を向上させます。 N = 3、* P &lt;0.05。

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+induces+fetal+type+II+cell+differentiation+via+an+epidermal+growth+factor+receptor-extracellular-regulated+protein+kinase+signaling+pathway.">Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway.</a> Am. J. Respir. Cell. Mol. Biol. 30, 76-83 (2004).</li><li>Wang, Y., Maciejewski, B. S., Lee, N., Silbert, O., McKnight, N. L., Frangos, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+fetal+type+II+epithelial+cell+differentiation+is+mediated+via+cAMP-PKA-dependent+signaling+pathway.">Strain-induced fetal type II epithelial cell differentiation is mediated via cAMP-PKA-dependent signaling pathway.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 291, L820-L827 (2006).</li><li>Sanchez-Esteban, J., Wang, Y., Filardo, E. J., Rubin, L. P., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+{beta}1,+{alpha}6,+and+{alpha}3+contribute+to+mechanical+strain-induced+differentiation+of+fetal+lung+type+II+epithelial+cells+via+distinct+mechanisms.">Integrins {beta}1, {alpha}6, and {alpha}3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 290, L343-L350 (2006).</li><li>Wang, Y., Maciejewski, B. S., Soto-Reyes, D., Lee, H. S., Warburton, D., Sanchez-Esteban, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+promotes+fetal+type+II+epithelial+cell+differentiation+via+shedding+of+HB-EGF+and+TGF-alpha.">Mechanical stretch promotes fetal type II epithelial cell differentiation via shedding of HB-EGF and TGF-alpha.</a> J. Physiol. 587, 1739-1753 (2009).</li><li>Wang, Y., Maciejewski, B. S., Drouillard, D., Santos, M., Hokenson, M. A., Hawwa, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+caveolin-1+in+mechanotransduction+of+fetal+type+II+epithelial+cells.">A role for caveolin-1 in mechanotransduction of fetal type II epithelial cells.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 298, L775-L783 (2010).</li><li>Lee, H. S., Wang, Y., Maciejewski, B. 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利害の衝突が宣言されません。

本稿では、我々は胎児のII型上皮細胞と線維芽細胞を分離し、Flexcellひずみ装置を使用して機械的な刺激にそれらを公開する方法について説明します。我々は、上皮細胞の分化1,2を評価し、受容体とストレッチ3月9日によって活性化シグナル伝達経路を研究するためにこのテクニックを使用しています。さらに、このメソッドはまた、機械的損傷10,11によって誘導され?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1A</td>
			<td>Sigma</td>
			<td>C9891</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screen cups</td>
			<td>Sigma</td>
			<td>CD1-1KT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Fisher Scientific</td>
			<td>09-754-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100 micron nylon mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-100-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 micron mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-30-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 micron mesh</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagen-1</td>
			<td>Collagen Biomed</td>
			<td>PC0701</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Sigma</td>
			<td>V-0132</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Elastin</td>
			<td>Sigma</td>
			<td>E-6402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bioflex plate</td>
			<td>Flexcell International</td>
			<td>BF-3001U</td>
			<td>Uncoated</td>
		</tr>
		<tr>
			<td>Flexcell Strain Unit</td>
			<td>Flexcell International</td>
			<td>FX-5000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an experimental system to study mechanotransduction in fetal lung cells

反復的な呼吸のような動きによって、流体の膨張によって子宮内で生成された機械的な力は、通常の肺の開発のために重要である。肺開発の主要なコンポーネントは、肺サーファクタントの主要な源、肺胞II型上皮細胞の分化である。これらの細胞はまた、肺胞腔、宿主防御、および損傷修復に体液の恒常性に参加しています。さらに、遠位の肺実質細胞が直接、出生後の機械的人工換気中の誇張されたストレッチを公開することができます。しかし、肺の細胞が肺の開発に影響を与えると肺傷害を促進するために機械的刺激を感知する正確な分子·細胞メカニズムは完全に理解されていません。ここでは、げっ歯類の胎児の肺からのII型細胞と線維芽細胞を単離するためのシンプルかつ高純度のメソッドを提供します。その後、我々は、機械的な力をシミュレートし、胎児の細胞に機械的な刺激を提供するためには、in vitroシステム、Flexcellひずみユニットで説明胎児肺の開発や肺損傷。この実験システムでは、ストレッチにさらされる胎児肺細胞では分子·細胞メカニズムを調べるための優れたツールを提供しています。このアプローチを使用して、私たちの研究室では、胎児の肺の開発と肺傷害にメカノに参加し、いくつかの受容体とシグナル伝達タンパク質を同定しました。

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An Experimental System to Study Mechanotransduction in Fetal Lung Cells

機械的な力は、肺の開発と肺傷害において重要な役割を果たしています。ここでは、げっ歯類の胎児の肺II型上皮細胞と線維芽細胞を分離するとを使用して機械的刺激にそれらを公開する方法について説明します。<em&gt; in vitroで</em&gt;システム。

胎児肺細胞におけるメカノトランスを研究するための実験システム

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

an-experimental-system-to-study-mechanotransduction-fetal-lung

Research

JoVE Journal

Bioengineering

12.9K ビュー.  Alpert Medical School of Brown University. 機械的な力は、肺の開発と肺傷害において重要な役割を果たしています。ここでは、げっ歯類の胎児の肺II型上皮細胞と線維芽細胞を分離するとを使用して機械的刺激にそれらを公開する方法について説明します。 in vitroでシステム。

ビデオ: An Experimental System to Study Mechanotransduction in Fetal Lung Cells

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

の軸索再生を研究するために、低予算レーザー軸索切断システムの構築 C.エレガンス</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

マルチチャンネルデュアル電界マイクロ流体チップを用いて、肺癌細胞の走電性の研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

脱細胞肺足場を使用してESC由来のマウス気道上皮細胞の生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

セミフレキシブル高分子を研究する多目的ツールとして DNA ナノチューブ

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

哺乳類細胞培養を均一な骨材を生産する O の形をした船で揺れ、軌道

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

ラットでの肝部分工学生体内での基盤として新しい手術手技

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Experimental Methods to Study Human Postural Control

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied by appropriate mathematical methods are needed to understand the role of the different sub-systems involved in human postural control. This article describes a protocol for performing perturbed standing experiments, acquiring experimental data, and carrying out the subsequent mathematical analysis, with the aim of understanding the role of musculoskeletal system and central control in human upright posture. The results generated by these methods are important, because they provide insight into the healthy balance control, form the basis for understanding the etiology of impaired balance in patients and the elderly, and aid in the design of interventions to improve postural control and stability. These methods can be used to study the role of somatosensory system, intrinsic stiffness of ankle joint, and visual system in postural control, and may also be extended to investigate the role of vestibular system. The methods are to be used in the case of an ankle strategy, where the body moves primarily about the ankle joint and is considered a single-link inverted pendulum.

This article presents an experimental/analytic framework to study human postural control. The protocol provides step-by-step procedures for performing standing experiments, measuring body kinematics and kinetics signals, and analyzing the results to provide insight into the mechanisms underlying human postural control.

人間の姿勢制御を研究する実験的方法

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...