著者らは、このモデルのためのインスピレーションのためのライナーSchultheissとヴォルフガングSchadenに感謝します。我々はまた、我々の研究を支援するために彼のすべての時間の多大な努力のためのキリスト教ドルフミュラーに感謝します。 私たちのアイデアを慎重に技術的な実現のためのロバートGöschlとハンスHoheneggerに感謝！

倫理的な許可患者の書面によるインフォームドコンセントを得た後、臍帯は、ヒト臍帯静脈内皮細胞（HUVEC）の単離のための婦人科における帝王切開から得た。許可はインスブルック医科大学（番号UN4435）の倫理委員会から与えられた。 1。IVSWTウォーターバスを準備します<ol><li>適切なタンクに水道水3.5リットルを準備します。水は37°C（プロトコル6を参照）に加熱する必要がある。 </li><li>水位が約3cmエッジ未満になるまで水槽に水を入れます。完全に水の膜をカバーするためにマインド。これは、約準備3.5 Lを必要とします</li><li>電源に温度センサーを接続します。 </li><li>水浴の後壁に意図されたフィッティングにセンサーを置く。 </li><li>温度センサの電源に加熱を接続します。なしでこれをしないでくださいヒーターなどのお風呂の水はプレキシグラスを溶かしてしまうだろう。 </li><li>水は37℃の安定した温度に達するまで待つ水浴を通して一定の温度を保証するために棒のいくつかの種類と定期的に水をかき混ぜる。 </li></ol> 2。細胞および培養フラスコを準備<ol><li>それらが所望の合流点に到達した直接までT25培養フラスコ、培養フラスコ中の細胞内で所望の密度で一晩種細胞。 </li><li>実験前、縦にフラスコに合わせます。 </li><li>右首下のまで、培養液で培養フラスコを埋める。汚染さになるだろう首や閉鎖キャップに接触している媒体としてフラスコ中にあまりにも多くのメディアを入れないでください。 </li><li>水からの汚染を防ぐために、フィルタなしに固体キャップをフラスコをねじ込み。槽内の水やお風呂自体は無菌ではないことに注意し。 </li><li>水浴中に挿入する前に、パラフィルムとキャップを封印。 </li> &lt;李&gt;提供スタンドに封入された培養フラスコを固定します。 </li><li>水槽に固定されたフラスコを挿入します。細胞培養フラスコの中央の衝撃波アプリケータのメンブレンの中央と同じ高さにあることを注意してください。バス側のラインがガイドします。 </li></ol> 3。治療パラメータを定義します。 <ol><li>衝撃波源と試料との間の距離を定義する。図6で説明したように、パイロット実験を求めるパラメータを行うことにより、最適な治療パラメータを識別する。 </li><li>衝撃波デバイス上の適切な治療パラメータ（エネルギー束密度、周波数）を選択する。再び、 図6を参照してください。 </li></ol> 4。衝撃波アプリケーション<ol><li>衝撃波アプリ上だけでなく、水浴の膜上、市販の超音波伝達ゲルをたっぷり量を置く。これは、カップリングを保証することである。空気や気泡は、アプリケーターと膜との間あってはならないそれは衝撃波を吸収する。 </li><li>膜に塗布器を接続し、膜の中心に安定して保持。それが水平に整列されていることに注意してください。 </li><li>水浴の内部プローブの正確な垂直方向の位置が示されたバスの側面にマーキングに沿ったものであることを確認してください。 </li><li>アプリケーターと水平線で安定フラスコの中心を持ってください。 </li><li>衝撃波デバイスをアクティブにして、全体の手順の間、安定した位置に培養フラスコなどのアプリケータを維持しながら、インパルスを適用します。 </li></ol> 5。後の処理<ol><li>水浴から培養フラスコを取る。 </li><li>一般的なペーパータオルでそれを乾燥させます。 </li><li>消毒剤を用いて正確にフラスコを拭きま​​す。 </li><li>パラフィルムシールとキャップを外します。 </li><li>遠心管にフラスコ内の媒質をピペット。 </li><li>適切に遠心します。遠心分離パラメータは、使用される細胞タイプに依存し、 <em&gt;例えば。 HUVECを、室温で290グラムを使用しています。 </li><li>フラスコに細胞培養培地を5mlの追加。細胞培養培地2mlで遠心分離し、細胞ペレットを再懸濁する。フラスコ内に懸濁物をピペットで。このように、治療中に切り離されている可能性があり、細胞断片または細胞が失われません。 </li></ol>落とし穴<ol><li>製剤ならびに治療は、細胞培養物の汚染を避けるために層流内側無菌条件下で行われるべきである。 </li><li>良好な細胞培養の練習は、プローブの汚染を避けることをお勧めします。特に、フラスコの外の消毒が強くバックインキュベーターにそれらを入れる前に、お勧めします。水浴だけでなく、内部の水は、無菌ではありません！ </li><li>水浴浴を建てたプレキシグラスの損傷を防ぐために水で満たされていない限り、電源にヒーターを接続しないでください。 </li><li>衝撃波APPLに超音波ゲルの寛大な量を使用浴に適切なカップリングと波動伝播を保証するicator。空気と小さな気泡が衝撃波を吸収するか！ </li><li>全体成長分野の治療を確保するために、アプリケータに関連した水浴の内部プローブの位置を確認してください。 </li><li>汚染を避けるためにあまりにも深く水にプローブをダンクしないでください。 </li></ol>

<ol>
	<li>Haupt, G., Haupt, A., Ekkernkamp, A., Gerety, B., Chvapil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+shock+waves+on+fracture+healing.">Influence of shock waves on fracture healing.</a> Urology. 39, 529-532 (1992).</li><li>Schaden, W., Fischer, A., Sailler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+shock+wave+therapy+of+nonunion+or+delayed+osseous+union.">Extracorporeal shock wave therapy of nonunion or delayed osseous union.</a> Clin. Orthop. Relat. Res. 387, 90-94 (2001).</li><li>Schaden, W., 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=Shock+wave+therapy+for+acute+and+chronic+soft+tissue+wounds:+a+feasibility+study.">Shock wave therapy for acute and chronic soft tissue wounds: a feasibility study.</a> J. Surg. Res. 143, 1-12 (2007).</li><li>Tepek&#246;yl&#252;, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+wave+treatment+induces+angiogenesis+and+mobilizes+endogenous+CD31/CD34-positive+endothelial+cells+in+a+hindlimb+ischemia+model:+Implications+for+angiogenesis+and+vasculogenesis.">Shock wave treatment induces angiogenesis and mobilizes endogenous CD31/CD34-positive endothelial cells in a hindlimb ischemia model: Implications for angiogenesis and vasculogenesis.</a> J. Thorac. Cardiovasc. Surg. 146, 971-978 (2013).</li><li>Nishida, 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=Extracorporeal+cardiac+shock+wave+therapy+markedly+ameliorates+ischemia-induced+myocardial+dysfunction+in+pigs+in+vivo.">Extracorporeal cardiac shock wave therapy markedly ameliorates ischemia-induced myocardial dysfunction in pigs in vivo.</a> Circulation. 110, 3055-3061 (2004).</li><li>Fukumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+cardiac+shock+wave+therapy+ameliorates+myocardial+ischemia+in+patients+with+severe+coronary+artery+disease.">Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease.</a> Coron. Artery Dis. 17, 63-70 (2006).</li><li>Gotte, G., Amelio, E., Russo, S., Marlinghaus, E., Musci, G., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-time+non-enzymatic+nitric+oxide+synthesis+from+L-arginine+and+hydrogen+peroxide+induced+by+shock+waves+treatment.">Short-time non-enzymatic nitric oxide synthesis from L-arginine and hydrogen peroxide induced by shock waves treatment.</a> FEBS Lett. 520, 153-155 (2002).</li><li>Wang, F. S., Wang, C. J., Huang, H. J., Chung, H., Chen, R. F., Yang, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+shock+wave+mediates+membrane+hyperpolarization+and+Ras+activation+for+osteogenesis+in+human+bone+marrow+stromal+cells.">Physical shock wave mediates membrane hyperpolarization and Ras activation for osteogenesis in human bone marrow stromal cells.</a> Biochem. Biophys. Res. Commun. 287, 648-655 (2001).</li><li>Mittermayr, R., 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=Extracorporeal+shock+wave+therapy+(ESWT)+minimizes+ischemic+tissue+necrosis+irrespective+of+application+time+and+promotes+tissue+revascularization+by+stimulating+angiogenesis.">Extracorporeal shock wave therapy (ESWT) minimizes ischemic tissue necrosis irrespective of application time and promotes tissue revascularization by stimulating angiogenesis.</a> Ann. Surg. 253, 1024-1032 (2011).</li><li>Baker, M., 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=Use+of+the+mouse+aortic+ring+assay+to+study+angiogenesis.">Use of the mouse aortic ring assay to study angiogenesis.</a> Nat. Protoc. 22, 89-104 (2011).</li></ol>

著者らは、開示することは何もありません。

インビトロ衝撃波治療のために提案されたモデルの意義は、波が既存のモデルとは対照的に、細胞培養物を通過した後に伝搬することができるという事実である。それによって、引張力などの物理的妨害の影響を回避することができる。モデルは、より密接に他の人が直接細胞培養フラスコに音波を印加することによってよりも、インビボ条件に似ている。 <p class="jove_content...

衝撃波は、例えば 、エネルギーの突然の解放に起因する音圧の波である。雷などの時に雷。医学では衝撃波が腎臓結石の崩壊のために砕石術において30年以上にわたって使用されてきた。 1980年代初頭における砕石術の患者における腸骨肥厚の偶発的所見ので、最初の研究は、骨の治癒1上の衝撃波処理（SWT）の効果を評価するために行った。長い骨の癒着不能の改善された癒しの印象的な結果は、2を観察することができた。その後、表示は軟部組織の傷3に拡大した。基礎研究の知見は、衝撃波は、任意の後続のダメージを与えることなく標的組織に生物学的刺激を引き起こすということを示した。血管新生増殖因子（ 例えば VEGF、PlGFは、FGF）の放出を有意血管形成が続く。これは、虚血性の病状に向けた指標の一層の拡大につながった。私たちのグループなどが、正EFFを示したECT動物モデルにおける虚血性心疾患に対するSWTのだけでなく、臨床試験4-6の。 しかし、SWTの物理的刺激が生体信号（メカノ）に変換されるかの正確なメカニズムは、大部分が未知のままである。継続的に薬が増加のいくつかの分野から、SWTの関心としては、メカニズムの探求はますます強烈になっている。したがって、 インビトロ衝撃波実験は重要性を増している。動物実験および費用対効果の低減に加えて、 インビトロ衝撃波処理（IVSWT）の最大の利点は、特定の細胞型の特定の挙動を研究する可能性があってもよい。最も可能性の高い治療された組織のすべての細胞が関与する衝撃波媒介組織再生においても、全身性の効果が記載されている。それにもかかわらず、各細胞型は、特定の役割を果たし、それ自身の固有の機能を有する。 IVSWTは、この特定の関数Aを検出することを可能にND、それによって私たちに複雑な基盤となるプロセスをよりよく理解することができます。 細胞培養物に対する衝撃波の影響についての今日の知識は、増殖の増加、細胞膜受容体の変化、増加および細胞分化の促進、成長因子および化学誘引物質の放出、ならびに増大した細胞遊走7-9を含む。 インビトロのモデルの中で最も邪魔で物理的効果、細胞培養物上に衝撃波を印加する様々な方法が記載されている。この事実は、細胞刺激の物理的条件は、これらのモデルの間でかなり異なっているように、それが結果を比較することは非常に困難であるという問題がある。一般的に、既存のすべてのモデルは、最高の細胞上に衝撃波を適用する方法に焦点を当てています。 しかし、この疑問が残っている：どのような細胞培養を通過した後の波はどうなりますか？主な問題は、音響の違い細胞培養培地と周囲空気のインピーダンスは、衝撃波の99％以上は、図1反射始めることが、高いことである。 による波が反射されるだけでなく、180°の位相シフトを細胞に強い引張力が発生し、その結果を図2つの媒体の音響インピーダンスの差である。 音響インピーダンスは、材料の密度と音速Z =ρX cは積として定義される。水の音響インピーダンスはZWater =144万NS / m 3であり 、空気がそれだけで420-S / M 3である。これら二つの値の差が大きい衝撃波の反射および位相シフトをもたらす。位相シフトは引張波に正圧力パルスをオンにします。 この引張力は、細胞に有害ではないとしても、 インビトロでインビボ衝撃波の効果を模倣するというアイデアに干渉する。 インビボこれら引張力はほとんど、大きな身体構造に起因し発生しません。 さらに、電波を実行し、バックにも入ってくるものを邪魔することができます。これは、干渉を引き起こす可能性があります。干渉の二つのタイプが知られている。建設的干渉は、両方の波が、それによって二倍の振幅を図3にその結果追加されていることを意味します。波は、直径方向に対向満たせば破壊的な干渉が発生する。これは、波の廃止（ 図3）を引き起こす。したがって、IVSWTは、細胞培養を通過した後、伝播する衝撃波を可能にモデルを必要とします。 IVSWT水浴上記の懸念は、次の考慮事項が記載された問題は、基本的には、衝撃波アプリケータのあらゆる種類を接続するために、膜を容器に構築されたプレキシグラスから構成されています。 図4を回避するための水浴を設計するために私たちを導く。この膜と塗布音波伝送GEとの間の結合のためのlが使用されなければならない。水浴は、ガスを水の中に固溶した場合に生じるキャビテーションを避けるために、脱気水で満たされている。制御ユニットに接続された温度センサと下部のヒーターは、 インビボ条件を模倣するための温度を調節するために、処置中にクールダウンするために細胞培養物を回避することができる。それは培養器で行われるように温度が摂氏37度で安定に保持することができる。細胞サンプルのためのホルダは、培養フラスコ又は管のいずれかの種類を浸漬することを可能にする。これにより、試料容器は、気泡が衝撃波をブロックするように完全培地で充填する必要がある！お風呂の後壁にくさび形の吸収体が順番に反射して干渉を回避するために戻って実行して取得しないように波を破棄。 他のIVSWTモデルに更なる利点は、アプリケータ及び培養フラスコとの間の距離を変化させる可能性がある。私たちのグループの調査結果との明らかに、このモデルを使用して他の人どのようにすべての細胞型が異なる治療パラメータに非常に特異的に反応する。これは衝撃波アプリケータの焦点に関して特定の位置にあると対照細胞することを可能として、さらに、波源と試料との間の距離を定義することが重要である。

<table border="0" cellpadding="0" cellspacing="0" width="2002">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Orthogold shock wave device</td>
			<td colspan="2" style="width:756px;">Tissue Regeneration Technologies, Woodstock, GA &#8211; manufactured by MTS-Europe GmbH, Konstanz, Germany</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">IVSWT Water Bath V2.0</td>
			<td style="width:537px;">Johann Hohenegger - Technical Products</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EBM-2 Basal Medium 500 m +EGM-2 SingleQuot Suppl.&#38;Growth Factors</td>
			<td style="width:537px;">Lonza</td>
			<td style="width:219px;">CC-3156 &#38; CC-4176</td>
			<td>This medium was used for the shown experiments with HUVECs to fill the cell culture flask. For other cell types, use the recommended medium.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"><a href="http://www.labplanet.com/pechiney-parafilm-m-pm996.html">Pechiney&#160;Parafilm M PM996</a></td>
			<td style="width:537px;">Pechiney Plastic Packaging</td>
			<td>PH-LF-PM996-EA at labplanet.com</td>
			<td>for sealing flasks</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Falcon Serological pipets 25ml</td>
			<td style="width:537px;">Becton Dickinson Labware</td>
			<td style="width:219px;">357525</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">CellMate II Serological Pipette&#160;</td>
			<td style="width:537px;">Matrix Technologies</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Skintact Ultrasonic Gel</td>
			<td style="width:537px;">Skintact</td>
			<td style="width:219px;">UL-01 250 ml</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">T25 cell culture flasks</td>
			<td style="width:537px;">COSTAR</td>
			<td style="width:219px;">3056</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">mikrozid disinfectant</td>
			<td style="width:537px;">Sch&#252;lke</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">3,5l degassed water</td>
			<td style="width:537px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">paper towels</td>
			<td style="width:537px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

shock wave application to cell cultures

衝撃波は、今日ではその再生効果によく知られている。基礎研究の知見は、衝撃波は、任意の後続の損傷なしに細胞または組織を標的とする生物学的刺激を引き起こすということを示した。従って、 インビトロ実験は、増大興味深い。細胞培養物上に衝撃波を印加する様々な方法が記載されている。一般的に、既存のすべてのモデルは、最高の細胞上に衝撃波を適用する方法に焦点を当てています。 しかし、この疑問が残っている：どのような細胞培養を通過した後の波はどうなりますか？細胞培養培地と周囲空気の音響インピーダンスの差は、衝撃波の99％以上が反射始めることが、高いことである！そこで我々は、主に波が細胞培養を通過した後、水中に伝播することを可能にするプレキシグラス構築された容器で構成されていたモデルを開発しました。これはキャビテーション効果だけでなく、そうでなければ、今後のものを乱す音波の反射を避けることができます。ウィスコンシン州このモデル番目の我々は、生体内条件を模倣し、それによって衝撃波の物理的刺激が生体細胞の信号（「メカノ」）に変換されます方法についてのより多くの知識を得ることができます。

我々は我々の臍帯から前述の単離されたヒト臍帯静脈内皮細胞（HUVEC）に衝撃波を印加記載の方法を用いて製造した。臍帯は、待機的帝王切開のセクションから入手した。 HUVECを電気油圧衝撃波療法システムとT25細胞培養フラスコ内で90％コンフルエンスで処理した。治療パラメータは、0.1ミリジュール/ mm 2で5ヘルツの周波数のエネルギー束密度であった。 300イ...

Watch this Scientific Journal Video about Shock Wave Application to Cell Cultures at JoVE.com

Shock Wave Application to Cell Cultures

衝撃波は、今日ではその再生効果によく知られている。従ってインビトロ実験は、関心の高まりである。そこで我々は、それによって気が散るの物理的効果を回避すること、生体内の状態に模倣することを可能にin vitroでの衝撃波試験（IVSWT）のためのモデルを開発しました。

細胞培養物衝撃波アプリケーション

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Research

Education

JoVE Journal

JoVE Core

Physics

Bioengineering

Unit 1

Waves

SCIENTISTS IN ACTION

12.6K ビュー.  Innsbruck Medical University. 衝撃波は、今日ではその再生効果によく知られている。従ってインビトロ実験は、関心の高まりである。そこで我々は、それによって気が散るの物理的効果を回避すること、生体内の状態に模倣することを可能にin vitroでの衝撃波試験（IVSWT）のためのモデルを開発しました。 

ビデオ: Shock Wave Application to Cell Cultures

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 ...

生物工学

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

共通液ハンドリングロボットでTruTipテクノロジーを使用して臨床検体からのDNAやRNAのハイスループット、自動抽出

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

エンジニアリングフィブリンベースの組織の筋線維芽細胞と制約の適用からの構築と細胞とコラーゲン（再）組織を誘導するためにひずみ

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

癌細胞浸潤への応用による腫瘍間質微小環境の組織工学

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

血管細胞リモデリングの研究への応用生理学的および病理学的血流のインビトロモデル

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

広域走査型プローブ ナノリソグラフィ自動配置と細胞培養研究用基板作製への応用

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

マイクロ流体チップを使用した決定論的方法で単一細胞ベースの共培養を確立する

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

生物学的に活性なサンゴリンマトリックスを用いた解離神経細胞培養の耐久性向上

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

癌細胞培養から採取した培地における液体クロマトグラフィー-質量分析法による選択キヌレニンの同時定量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...