著者らは本研究で使用したブタ血清を提供するための画像処理チタンのセクションとジェミニバイオ製品に関する貴重なアドバイスのためにライカマイクロシステムズに感謝します。我々はまた、RC1HL099863 - 01&quot;デバイスを補助循環の生体適合性を向上させるために自家EPCライニング&quot;グラントを通じて、支援のためにNIHに感謝したい。さらに、我々は、アレクサンドラジャンセンの国立科学財団大学院研究フェローシップの支援に感謝しています。我々はまた、外科手術と私たちの研究動物の処理の多くの側面で支援するためにジョージクイック、マイクロウとIanthiaパーカーを感謝したい。スティーブンオーウェンは、私たちのシードデバイスの機械加工の多くの部分で非常に貴重なものとチタン管を切断している。

<ol>
	<li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+bleeding+and+clotting+in+the+cardiac+surgery+patient:+from+vascular+endothelium+to+circulatory+assist+device+surface.">Pathophysiology of bleeding and clotting in the cardiac surgery patient: from vascular endothelium to circulatory assist device surface.</a> Circulation. 122, 2068-2077 (2010).</li><li>Achneck, H. E., Sileshi, B., Lawson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+biology+of+bleeding+and+clotting+in+the+surgical+patient.">Review of the biology of bleeding and clotting in the surgical patient.</a> Vascular. 16, 6-13 (2008).</li><li>Arvidsson, S., Askendal, A., Tengvall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+plasma+contact+activation+on+silicon,+titanium+and+aluminium.">Blood plasma contact activation on silicon, titanium and aluminium.</a> Biomaterials. 28, 1346-1354 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+titanium+cardiovascular+devices+seeded+with+autologous+blood-derived+endothelial+progenitor+cells:+EPC-seeded+antithrombotic+Ti+Implants.">The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants.</a> Biomaterials. 32, 10-18 (2011).</li><li>Kang, C., Bonneau, M., Brouland, J. P., Bal dit Sollier, C., Drouet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+pig+models+of+venous+thrombosis+mimicking+human+disease.">In vivo pig models of venous thrombosis mimicking human disease.</a> Thromb Haemost. 89, 256-263 (2003).</li><li>Ueberrueck, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+ovine+and+porcine+animal+models+for+biocompatibility+testing+of+vascular+prostheses.">Comparison of the ovine and porcine animal models for biocompatibility testing of vascular prostheses.</a> J Surg Res. 124, 305-311 (2005).</li><li>Velik-Salchner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+values+for+thrombelastography+(ROTEM)+and+selected+coagulation+parameters+in+porcine+blood.">Normal values for thrombelastography (ROTEM) and selected coagulation parameters in porcine blood.</a> Thromb Res. 117, 597-602 (2006).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Heart Association Scientific Sessions, Abstract Oral Sessions, Medical Aspects End Stage Heart Failure: Transplantation and Device Therapies. , (2010).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+titanium+ventricular+assist+device+surfaces+after+gold/palladium+coating+for+scanning+electron+microscopy.">Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy.</a> Microsc Res Tech. 73, 71-76 (2010).</li><li>Ford, J. W., Welling, T. H., Stanley, J. C., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKH26+and+125I-PKH95:+characterization+and+efficacy+as+labels+for+in+vitro+and+in+vivo+endothelial+cell+localization+and+tracking.">PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking.</a> J Surg Res. 62, 23-28 (1996).</li></ol>

利害の衝突は宣言されません。

ここで提示細胞播種チタンチューブの方法は、迅速かつ均一endothelializeに医師や科学者を可能に血液を接触させ、移植可能なデバイスの表面を。我々は末梢血サンプルからEPCを分離し、拡大しているので、大きな侵襲的処置は、これらの細胞を回収する必要はありません。また、EPCは自己であるため、セルシードのインプラントへの免疫反応の危険性が除去されます。彼のプロトコルで示さ原則は、チタン管のためだけでなく、心血管医学に利用されている他の多くの生体材料、のために利用することができます。 我々は汚染物質妥協の細胞接着のそのあらゆる接着フィルムを見つけたとして、このプロトコルの重要なステップは、チタン管の念入りな清掃です。さらに、遅い回転速度が（チューブの直径に反比例）播種の過程で不可欠である、そのようなことEPCは徐々に落ち着くとTiのチューブが移動しているように表面に付着することがあります。 注入直前に播種の我々の手法は非現実的なex vivoで培養時間を避けることができます。細胞が個々に付着し、後ですぐに流れの再構築は、次のシートとして塞栓の可能性を回避すること、 生体内でコンフルエントシートを形成する。私たちの先行研究では、EPCはコンフルエント層に成長した後、彼らはさらに内皮シートのすべての可能な脱落を最小限に抑え、彼らはしっかりと付着する細胞外マトリックスを作ることを示している。塞栓剥離の可能性を完全に排除できないものの、それは、デバイス全体の表面の血栓症より多種多様のリスクが低く、この治療法は防ぐように設計されている問題のようです。 低せん断への移植の我々のアプローチは、下大静脈の血栓環境では、血液適合性および装置5,6の血栓症を研究するための最も信頼される大規模な動物モデルのいずれかを利用しています。すべての動物の世話と実験は実験動物の管理と利用のための健康ガイドラインの国立研究所ととのみデューク大学動物実験用の委員会の承認後にしたがって実施されたことに注意してください。 成功した生体材料およびデバイスをテストするには、この注入法を利用するために、それは辛抱強くIVCのセグメントを骸骨になると、デバイスの周囲の出血が&quot;偽腔&quot;に由来していないようにデバイスを挿入するための準備として、すべての静脈の分岐血管を、連結することが重要です。別の重要なステップは、内側のチューブの表面上に細胞がveinotomyの閉鎖中に湿ったままで、再灌流が開始される前にいるようなデバイスの内腔へのDPBSの追加です。デバイスは、それが移植された場所に見つからない場合は、それはIVCの&quot;上流&quot;に移行している可能性があります。 PVCチューブの3 mmのセクションをチューブがしっかりとその現在の場所に固定されるように - これは、静脈の壁を通って、2を介して縫合糸（4 - O Prolene）を配置することによって防ぐことができます。研究者はそうでなければ特許チューブに植後の図8に示すように蛍光あらかじめ標識された細胞を見つけることが困難なはずですが、これは細胞が首尾一貫したシートとして剥離れている可能性があります。これは、チューブと一緒に静脈を固定した後、3 Tiのチューブセクションの周囲の静脈や放電アセンブリの非常に穏やかな解離することによって防ぐことができます。 当社の技術はEPC -播種によって心血管装置の血栓症を防止するための証明の概念を提供します。この技術は、患者自身の血管内皮前駆細胞が並ぶ&quot;生体&quot;インプラントの開発に使用することができます。私たちのブタ動物モデルでの実現可能性調査は、臨床の現場にこの&quot;オーダーメイド医療&quot;の翻訳に向けた最初のステップのために用意されています。

<table border="1"><tbody><tr><td> 試薬 </td><td> 企業名 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>アセプロマジン</td><td>ベーリンガーインゲルハイム</td><td> BIC670025 </td><td> NAC＃10280002 </td></tr><tr><td> Alconoxパワード精密クリーナー</td><td> Alconox </td><td> 1104 </td><td></td></tr><tr><td>バルフォア外科トラクター</td><td>アドラー</td><td> N / </td><td></td></tr><tr><td> Baytril </td><td>バイエル</td><td> APVMA 46028/0705 </td><td></td></tr><tr><td>翼状針（19G） </td><td>テルモ</td><td> SV19CLK </td><td></td></tr><tr><td>クロルヘキシジン</td><td> 3M </td><td> 9200 </td><td></td></tr><tr><td>クランプ（45度） </td><td> Aesculap </td><td> FC339T </td><td></td></tr><tr><td> DPBS (-/-) </td><td>ギブコ</td><td> 14190-144 </td><td></td></tr><tr><td> DPBS (+/+) </td><td>ギブコ</td><td> 14040-133 </td><td></td></tr><tr><td> DuraPrep </td><td> 3M </td><td> 8635 </td><td></td></tr><tr><td> EBM - 2培地</td><td>ロンザ</td><td> CC - 3156 </td><td>両方の血清自由かつ完全な増殖培地のベース</td></tr><tr><td> EGM - 2 SingleQuots </td><td>ロンザ</td><td> CC - 3162 </td><td>両方の血清自由かつ完全な増殖培地用EBM - 2と組み合わせて使用</td></tr><tr><td>電気焼灼ツール</td><td> Valleylab </td><td> SurgII - 20 </td><td></td></tr><tr><td>エメリー布</td><td> 3M </td><td> 60-0700-0425-8 </td><td> 240グリット</td></tr><tr><td> Euthasolの安楽死のソリューション</td><td> Virbackアニマルヘルス</td><td></td><td>穴田＃：200〜071 </td></tr><tr><td>フェンタニルパッチ</td><td> Actavis </td><td></td><td> NDC＃67767-120-18 </td></tr><tr><td>フルニキシン</td><td>シェリングプラウ</td><td></td><td> NAC＃：10470183 </td></tr><tr><td>フォーリーカテーテル（16F） </td><td>吟遊詩人</td><td> 730116 </td><td></td></tr><tr><td> HBSS </td><td>シグマ</td><td> H8264 - 500ML </td><td></td></tr><tr><td>ヒートガン</td><td>ミルウォーキー</td><td> 8988〜20 </td><td></td></tr><tr><td>ヘパリン</td><td></td><td></td><td> NDC＃：25021 </td></tr><tr><td> Histopaque - 1077 </td><td>シグマ</td><td> H8889 - 500ML </td><td></td></tr><tr><td>挿管チューブ</td><td> Mallinckrodt </td><td> 86113 </td><td></td></tr><tr><td>イソフルレン</td><td> MWI、メリディアン</td><td></td><td> NDC＃13985-030 </td></tr><tr><td>静脈カテーテル（18G） </td><td>ベクトンディッキンソン- </td><td> 381547 </td><td></td></tr><tr><td>ケタミン</td><td>フォートドッジ</td><td></td><td> NDC＃0856-2013 </td></tr><tr><td>レベル</td><td>スワンソン</td><td> LLA001 </td><td></td></tr><tr><td>ルアーロック先端キャップ</td><td> CMLサプライ</td><td> 909から001 </td><td></td></tr><tr><td> Marcaine </td><td>ホスピーラ</td><td></td><td> NDC＃：0409-1560-10 </td></tr><tr><td> Metzenbaumはさみ</td><td>アドラー</td><td> N / </td><td></td></tr><tr><td>マイクロイントロ（5F） </td><td>ゴールト</td><td> KIT 002から01 </td><td></td></tr><tr><td>モスキート止血鉗子</td><td>アドラー</td><td> N / </td><td></td></tr><tr><td>モーター</td><td> HerbachとRademan </td><td> H1 - 08 </td><td></td></tr><tr><td>オキシモルホン</td><td>遠藤研究所</td><td></td><td> NDC：63481-624-10 </td></tr><tr><td> PKH26色素キット</td><td>シグマ</td><td> PKH26GL - 1KT </td><td></td></tr><tr><td>ブタ血清</td><td>ジェミニバイオ製品</td><td> 100から115 </td><td>完全増殖培地で2％濃度</td></tr><tr><td>ポッツはさみ</td><td>アドラー</td><td> N / </td><td></td></tr><tr><td>正確なビスタスキンステープラー</td><td> 3M </td><td> 3998 </td><td></td></tr><tr><td> PVCチューブ</td><td> McMaster-Carr/Insultab </td><td> 7132K117 </td><td>拡大ID 15.88ミリメートル、 回収されたID 7.95ミリメートル</td></tr><tr><td>アングルミディアムサイズ</td><td>アドラー</td><td> N / </td><td></td></tr><tr><td>手術用メスの刃（＃10-15） </td><td>吟遊詩人</td><td> 373910 </td><td></td></tr><tr><td>シリコンチューブ</td><td>マクマスター - カー</td><td> 51735K26 </td><td> 16.64ミリメートルOD、9.52ミリメートルのID </td></tr><tr><td>シリンジ（5ccの） </td><td>ベクトン-ディッキンソン（BD） </td><td> 309603 </td><td></td></tr><tr><td> Tegaderm </td><td> 3M </td><td> 90001 </td><td></td></tr><tr><td>三方活栓</td><td>ケンドール</td><td> 170060 </td><td></td></tr><tr><td>チタンチューブ</td><td>ティコチタン</td><td> N / </td><td> ½&quot;OD、0.065&quot;壁、0.370&quot;ID、0.1737ポンド/フィートのように指定</td></tr><tr><td>トリプシン</td><td>ロンザ</td><td> CC - 5012 </td><td></td></tr><tr><td>トリプシン中和溶液（TNS） </td><td>ロンザ</td><td> CC - 5002 </td><td> 0.03パーセント</td></tr><tr><td> Vetropolycin </td><td> Pharmadermアニマルヘルス</td><td></td><td> NAC＃：12920110 </td></tr><tr><td> Vicryl縫合糸（3-0） </td><td>エチコン</td><td> J808T </td><td></td></tr><tr><td>水浴ソニケーター</td><td>ブランソン</td><td> B200 </td><td></td></tr></tbody></table>

autologous endothelial progenitor cell-seeding technology and biocompatibility testing for cardiovascular devices in large animal model

植込み型心血管系デバイスは、血栓の形成1,2,3のリスクをもたらす人工材料（例えばチタン（Ti）、ポリテトラフルオロエチレン拡大）、から製造されています。我々は、自己血由来のヒトまたはブタ血管内皮前駆細胞（EPC）4にチタンチューブの内側表面を整列する方法を開発した。豚の下大静脈（IVC）においてブタのEPCのコンフルエント層を含むチタンチューブを注入することにより、我々は、大規模なモデル動物の血栓環境における細胞播種面の改善された生体適合性をテストし、変更されていない裸の金属表面とそれを比較5,6,7（ 図1）。このメソッドは、注入から数分以内にデバイスをendothelialize および in vivo でその抗血栓機能をテストするために使用することができます。 末梢血は、50キロヨークシャー豚とEPCは4,8を分離するために培養したその単核細胞分画から得られた。チタンチューブ（9.4 mmのID）には3つの4.5センチメートル縦断面にあらかじめカットし、熱収縮チューブで再組み立てされた。シードデバイスは、チタン管の遅い回転を可能になる、建設された。 我々は、豚に開腹術を行い、腸や膀胱を外部化。シャープと鈍的切開は近右腎動脈の遠位の分岐からIVCを骸骨にするために使用されていました。チタンチューブは、均一な細胞コーティング9を達成するために10 RPH × 30分でEPCサスペンションと回転自家蛍光標識に満ちていた。 100 USP / kgのヘパリンの投与後、IVCと腰静脈の両端をクランプした。 4cmのveinotomyが行われ、デバイスが挿入され、リン酸緩衝生理食塩水で満たされた。 veinotomyが4から0 Prolene実行されている縫合糸で閉じていたとして、あるクランプは解除空気IVCに削除されました。プロシージャの最後で、筋膜が0 - PDS（ポリジオキサノン縫合）で近似された、皮下のスペースは2-0 Vicrylで閉じ、皮膚が閉じホチキス。 3の後に - 21日、豚は、専用バスブロックと固定を植デバイス安楽死させた。チタンチューブは分解し、内部表面は、蛍光顕微鏡で画像化した。 我々は、EPC -シードチューブが特許残ったのに対し、裸の金属Ti管が完全に閉塞ことがわかった。さらに、我々は内部の血液接触面にEPCのコンフルエントな層を示すことができた。 おわり、私たちの技術は、デバイスの血栓症を防ぐために、自家EPCSに注入から数分以内にチタンチューブをendothelializeするために使用することができます。私たちの外科的方法は、最小限の失血とEPC -シードの表面の中断はそのような変更されたデバイスの改良された生体適合性をテストすることができます。

Watch this Scientific Journal Video about Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model at JoVE.com

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

自己細胞とテスト生体適合性とチタンの血液接触生体材料をシードする方法について説明する。このメソッドは、ブタ静脈cavaeに外科的に移植後数分以内に接種した内皮前駆細胞およびチタンチューブを、使用しています。この手法は、他の多くの埋め込み型医用機器に適応可能です。

大動物モデルにおける心血管デバイス用自家血管内皮前駆細胞播種技術と生体適合性試験

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

autologous-endothelial-progenitor-cell-seeding-technology

Nanyang Technological University

Research

JoVE Journal

Bioengineering

20.3K ビュー.  Duke University. 自己細胞とテスト生体適合性とチタンの血液接触生体材料をシードする方法について説明する。このメソッドは、ブタ静脈cavaeに外科的に移植後数分以内に接種した内皮前駆細胞およびチタンチューブを、使用しています。この手法は、他の多くの埋め込み型医用機器に適応可能です。

ビデオ: Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

使用プラ​​センタアクロス異物およびナノ材料の輸送速度の決定<em&gt; ex vivoで</em&gt;ヒト胎盤灌流モデル

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

生物工学

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

内皮増殖、バリアー機能、運動性の定量のための電気細胞 - 基質インピーダンスセンシング

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

有毒吸入化学試験のためのin vitroでの細胞培養モデルにおける

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Microtissue安定化のためのマイクロ流体デバイスの層ごとのコラーゲン沈着

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

内皮細胞捕捉および保持のための強磁性ベアメタルステント

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

細胞播種用カスタム Agarose 井戸とティッシュ リング自己組織型の 3 D プリントを使用しての作製

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

発芽血管を研究するためのテッセレーション足場の段階的な細胞播種

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

生体組織の刺激のための電界・磁場装置

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

肺高血圧症と右心室不全の大型動物モデル:羊の左肺動脈結紮と進行性主肺動脈バンディング

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.