Name: creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting
Uploaded: 2017-07-02T13:00:00.000+00:00
Duration: 4 min 40 s
Description: Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

著者らは、心臓血管研究のための基金基金とメリーランド幹細胞研究基金（2016-MSCRFI-2735）という資金源を認めています。

1.心筋細胞の調製<ol><li>記載されているように、基底膜マトリックスでコーティングされた6ウェルプレート上で、ヒト誘導性多能性幹細胞（hiPSC）を生成および培養する。10 。 </li><li>以前に記載された方法11,12を用いてhiPSCをhiPSC由来心筋細胞（hiPSC-CM）に分化させる。 </li><li>分化後19日目に、室温で5分間、各ウェルで2mLのトリプシン/ EDTA 0.05％を用いて心筋細胞を単離する。 </li><li>心筋細胞を光学顕微鏡下でモニターし、細胞の解離を観察する。 </li><li> 2mLのトリプシンインヒビター0.0125％を用いてトリプシンを中和する。 </li><li>単離された心筋細胞をプールし、モーター付きピペットフィラーを使用して1つの50mLコニカルチューブに移す。 </li><li>室温で250xgで5分間細胞懸濁液を遠心分離してペレットを得る。 </li><li>ペレットをロズウェルパーク5mLに再懸濁するB-27（RPMI / B-27細胞培地）を補充したMemorial Institute（RPMI）細胞培地。 </li><li>細胞培地に20μLの細胞をピペットで入れ、同量の0.4％Trypan Blue溶液で染色します。 </li><li>自動細胞計数器または手動血球計算器を使用して、細胞懸濁液の濃度および細胞生存率を数え、得る。 </li></ol>繊維芽細胞の調製<ol><li>ヒト心臓線維芽細胞（HCF）（成人心室型）細胞系を開始する。 </li><li>継代し、HCF培養プロトコールに従ってそれらを単離する。 </li><li>細胞培地中の20μLの細胞をピペットで等量のTrypan Blue溶液0.4％で染色する。 </li><li>新しい細胞懸濁液の濃度および細胞生存率を数え、得るために、自動細胞カウンターまたは手動血球計算機を使用する。 </li></ol> 3.内皮細胞の調製<ol><li>人間のumbを開始する記載されているように、静脈内皮細胞（HUVEC）系統14 。 HUVEC培養プロトコールに従い、それらを単離する（ 14） 。 </li><li>細胞培地に20μLの細胞をピペットで入れ、同量の0.4％Trypan Blue溶液で染色します。 </li><li>自動細胞計数器または手動血球計算器を使用して、細胞懸濁液の濃度および細胞生存率を数え、得る。 </li></ol> 4.共培養： <ol><li> RPMI / B-27細胞培地中で3種類の細胞型（hiPSC-CM、HCF、およびHUVEC）を混合して、hiPSC-CM：HCF：HUVEC細胞比50mLコニカルチューブに混合細胞溶液のストックを生成する。 70:15:15およびmL当たり165,000細胞の濃度。 </li><li>混合細胞溶液を、マルチチャネルピペットを使用してウェル当たり200μLまたは1ウェルあたり33,000細胞の超低付着96ウェルU底プレートに分配する。 </li><li> 96ウェルプレートを3日間インキュベートする（37℃、5％の炭素二酸化炭素湿度95％）。 </li><li>光学顕微鏡下で個々のウェルの中央と底に混合細胞多細胞スフェロイドの存在を検査する。 注：2次元の顕微鏡投影では、これらの回転楕円体は円形に見えます （ 図1 ）。スフェロイドは、細胞播種後、24時間以内に極低温付着96ウェルプレートに形成されるべきである。スフェロイドは96ウェルプレートに細胞を播種した後、48時間以内に鼓動を開始するはずです。鼓動していないスフェロイドは、最終的な3Dバイオプリントされた心臓パッチが機能することを保証するために3Dバイオプリントに使用すべきではありません。コネキシン43（Cx43）およびトロポニンのスフェロイド免疫蛍光法を用いて、球状細胞の組成と品質を評価することができます15 。 </li></ol> 5.足場のない心臓組織の3Dバイオプリント<ol><li>スフェロイドを含むプレートを、足場のない回転楕円体ベースの3Dバイオプリンタのマガジンにロードします。 </li><li> 3Dバイオプリンタの電源を入れ、3Dバイオプリントソフトウェアを実行します。 <ol><li> 「補助」、「操作準備」、「検査モード」、「原点復帰」の順にクリックします。 </li><li> 「スタート」の下にある緑色のアイコンをクリックして回転楕円体検査を開始します。 </li><li>画面上の &quot;（2）Dia（μm）&quot;欄の下に回転楕円体の直径が表示されていることを確認します。 </li></ol></li><li>平均回転楕円体の直径を100μmに近づけるには、「初期設定」をクリックし、平均回転楕円体直径をStack Zピッチ（μm）に入力します。 </li><li> 「合計」をクリックすると、「レイヤー」の下の数字が自動的に再調整されます。 [適用]をクリックして設定を確定します。 </li><li> 「ニードルアレイマップ」をクリックし、「レイヤー番号」に移動して、3Dバイオプリント用に必要なレイヤーを選択し、個々の回転楕円体の位置を選択して、画面の左側のマップに目的の3Dバイオプリントデザインを描画します。 Click「適用」をクリックして3Dバイオプリントデザインを確認します。 </li><li> 「ニードルアレイチェック」をクリックしてニードルアレイ検査を開始します。ニードルアレイにピントが合っていない場合は、0〜500μmの範囲の値を &quot;OffsetZ（μm）&quot;に入力して、500μmから100μmまで減少するようにカメラのフォーカスを調整します。 </li><li> 「検査パラメータ」、「タイプ1」をクリックし、「直径（μm）」を希望の直径範囲に設定します。 「真円度（％）」と「平滑度（％）」を希望のパーセンテージに設定します。 [適用]をクリックして設定を確定します。 注：ここで回転楕円体の直径は450μm〜550μmに設定されています。 「真円度（％）」および「滑らかさ（％）」は、個々の回転楕円体のコンピュータ計算による評価です。ここでは、「真円度（％）」は60％、「平滑度（％）」は70％です。 </li><li> 「存在」をクリックし、「直径（μm）」を同じ設定に更新します前の手順では、 [適用]をクリックして設定を確定します。 </li><li> 「Stack Mode」をクリックし、「Start」の下にある緑色のアイコンをクリックして、3Dバイオプリンタのノズルを真空吸引し、針アレイの特定の位置にスフェロイドを配置して、回転楕円体のピックアップを開始します。 注：81個の回転楕円体（9個の回転楕円体と9個の回転楕円体）からなる心臓パッチの場合、3Dバイオプリントには約20〜30分かかります。 </li><li>バイオプリント後、滅菌ピンセットを使用して、3Dバイオプリントパッチを含む針アレイをピックアップし、150mLのRPMI / B-27細胞培地を含む250mL滅菌ビーカーに移す。 </li><li> 3日間（37℃、5％二酸化炭素、95％湿度）、3Dバイオプリントパッチを針アレイとインキュベートする。 </li></ol> 6.ニードルアレイからの3D Bioprintedパッチの除去とパッチ成熟<ol><li> 3日間のインキュベーションの後、一方の手は針アレイの基部に、他方のものはプラスチックカバー上のt貼付し、プラスチック製のカバーを静かにスライドさせて、3Dバイオプリントパッチをニードルアレイから外す。 注意：リン酸緩衝生理食塩水（PBS）による潤滑は、必要に応じて、このステップの直前のニードルに適用して、スムーズで簡単に取り外すことができます。 </li><li>一対の無菌鉗子を使用して、その上に3Dバイオプリントパッチを入れたプラスチックカバーをピックアップし、パッチとプラスチックカバーをRPMI / B-27細胞培地を含む35mmディッシュに移す。 </li><li>ピンセットをプラスチックカバーに固定した状態で、RPMI / B-27細胞培地のパッチでプラスチックカバーを静かに振って、パッチを培地に放出させます。 </li><li> 3Dバイオプリントされたパッチが光学的に検査され、3Dバイオプリントされたパッチが機能しているかどうかを確認しているかどうかを視覚化する。 </li><li> 3日以上（37℃、5％二酸化炭素、95％湿度）、35mmディッシュ内の3Dバイオプリントパッチをインキュベートする。 注：3日後、回転楕円体が融合し、針があったパッチはもはや見えなくなるはずです。 </li></ol>

<ol>
	<li>Murphy, S. V., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+tissues+and+organs.">3D bioprinting of tissues and organs.</a> Nat Biotech. 32, 773-785 (2014).</li><li>Bajaj, P., Schweller, R. M., Khademhosseini, A., West, J. L., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+biofabrication+strategies+for+tissue+engineering+and+regenerative+medicine.">3D biofabrication strategies for tissue engineering and regenerative medicine.</a> Ann Rev Biomed Eng. 16, 247-276 (2014).</li><li>Patra, S., Young, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+3D+Printing+Techniques+and+the+Future+in+Biofabrication+of+Bioprinted+Tissue.">A Review of 3D Printing Techniques and the Future in Biofabrication of Bioprinted Tissue.</a> Cell Biochem Biophy. 74, 93-98 (2016).</li><li>Moldovan, N. I., Hibino, N., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+the+Kenzan+Method+for+Robotic+Cell+Spheroid-Based+Three-Dimensional+Bioprinting.">Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting.</a> Tissue Eng Part B Rev. , (2017).</li><li>Cui, X., Boland, T., D'Lima, D. D., Lotz, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+inkjet+printing+in+tissue+engineering+and+regenerative+medicine.">Thermal inkjet printing in tissue engineering and regenerative medicine.</a> Recent Pat Drug Deliv Formul. 6, 149-155 (2012).</li><li>Murphy, C. M., Haugh, M. G., O'Brien, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+mean+pore+size+on+cell+attachment,+proliferation+and+migration+in+collagen-glycosaminoglycan+scaffolds+for+bone+tissue+engineering.">The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering.</a> Biomater. 31, 461-466 (2010).</li><li>Noguchi, 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=Development+of+a+three-dimensional+pre-vascularized+scaffold-free+contractile+cardiac+patch+for+treating+heart+disease.">Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease.</a> J Heart Lung Transpl. 35, 137-145 (2016).</li><li>Mironov, V., 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=Organ+printing:+tissue+spheroids+as+building+blocks.">Organ printing: tissue spheroids as building blocks.</a> Biomater. 30, 2164-2174 (2009).</li><li>Itoh, 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=Scaffold-Free+Tubular+Tissues+Created+by+a+Bio-3D+Printer+Undergo+Remodeling+and+Endothelialization+when+Implanted+in+Rat+Aortae.">Scaffold-Free Tubular Tissues Created by a Bio-3D Printer Undergo Remodeling and Endothelialization when Implanted in Rat Aortae.</a> PloS One. 10, e0136681 (2015).</li><li>Lieu, P. T., Fontes, A., Vemuri, M. C., Macarthur, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+with+CytoTune,+a+non-integrating+Sendai+virus.">Generation of induced pluripotent stem cells with CytoTune, a non-integrating Sendai virus.</a> Methods Mol Biol. 997, 45-56 (2013).</li><li>Li, S., Cheng, H., Tomaselli, G. F., Li, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+basis+of+excitation-contraction+coupling+in+human+pluripotent+stem+cell-derived+ventricular+cardiomyocytes+revealed+by+Ca2++spark+characteristics:+direct+evidence+of+functional+Ca2+-induced+Ca2++release.">Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell-derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics: direct evidence of functional Ca2+-induced Ca2+ release.</a> Heart Rhythm. 11, 133-140 (2014).</li><li>Boheler, K. 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=A+Human+Pluripotent+Stem+Cell+Surface+N-Glycoproteome+Resource+Reveals+Markers,+Extracellular+Epitopes,+and+Drug+Targets.">A Human Pluripotent Stem Cell Surface N-Glycoproteome Resource Reveals Markers, Extracellular Epitopes, and Drug Targets.</a> Stem Cell Rep. 3, 185-203 (2014).</li><li>ScienCell Research Laboratories. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cardiac Fibroblasts (adult ventricular) Product Sheet. , (2017).</li><li>Lonza Walkersville, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Endothelial Cell Systems - Technical Information &amp; Instructions.,. , (2015).</li><li>Thermo Fisher Scientific, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunofluorescence Method for IHC Detection. , (2017).</li><li>Murata, D., 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=A+preliminary+study+of+osteochondral+regeneration+using+a+scaffold-free+three-dimensional+construct+of+porcine+adipose+tissue-derived+mesenchymal+stem+cells.">A preliminary study of osteochondral regeneration using a scaffold-free three-dimensional construct of porcine adipose tissue-derived mesenchymal stem cells.</a> J Orthop Surg Res. 10, (2015).</li><li>Mosadegh, B., Xiong, G., Dunham, S., Min, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+progress+in+3D+printing+for+cardiovascular+tissue+engineering.">Current progress in 3D printing for cardiovascular tissue engineering.</a> Biomed Mater. 10, 034002 (2015).</li><li>Chang, C. C., Boland, E. D., Williams, S. K., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-write+Bioprinting+Three-Dimensional+Biohybrid+Systems+for+Future+Regenerative+Therapies.">Direct-write Bioprinting Three-Dimensional Biohybrid Systems for Future Regenerative Therapies.</a> J Biomed Mater Res. Part B, Appl Biomater. 98, 160-170 (2011).</li><li>Lee, J. M., Sing, S. L., Tan, E. Y. S., Yeong, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+in+cardiovascular+tissue+engineering:+a+review.">Bioprinting in cardiovascular tissue engineering: a review.</a> International J Bioprinting. 2 (2016), (2016).</li></ol>

著者は何も開示することはない。

It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histolog...

3Dバイオプリント1,2,3の方法にはさまざまなものがあります。 3Dバイオプリントは、インクジェットバイオプリンティング、マイクロ押出バイオプリント、レーザーアシストバイオプリント、方法の組み合わせ、またはより新しいアプローチなどの例を用いて、印刷技術1によって分類されることが多い。 3Dバイオプリントは、スキャフォールドフリーまたはスキャフォールド依存法に分類することもできます4 。 3Dバイオプリントのほとんどの方法は、生体材料、 例えばバイオインキ5または足場6が必要な足場依存性である。しかしながら、足場依存性3Dバイオプリンティングは、足場材料の免疫原性、独自のバイオインキのコスト、分解産物の遅い速度および毒性など、多くの問題および限界に直面している4,7。 Scafスフェロイドを用いた無折返し心臓組織工学が試​​みられており、足場依存性組織工学のこれらの欠点を克服する可能性がある。しかし、この論文の著者が認めたように、生物工学の過程で、固定された場所でスフェロイドをしっかりと取り扱い、配置することは困難でした。 3Dバイオプリントと回転楕円体ベースの組織工学の併用は、これらの困難を克服する可能性を秘めています。このプロトコールでは、スフェロイドの形態の細胞のみを用いて、他の生体材料を用いずに心臓組織の3Dバイオプリントを説明する。 足場のない回転楕円体ベースの3Dバイオプリンタ9は、真空吸引を使用して個々の回転楕円体をピックアップし、それらを針アレイ上に配置する能力を有する。 3次元バイオプリンティングにおける針アレイ上のスフェロイドの位置づけの概念は、古代ジャパ（ Japa ）においてニードルアレイ（「 ケンザン （ kenzan ）」として知られている）花の配置、 生け花の nese芸術。このシステムは、スフェロイドを任意の構成に正確に配置することを可能にし、個々のスフェロイドを短時間で融合させて、3Dバイオプリント組織を作製する。したがって、この方法は、スフェロイドを容易に操作することを可能にし、足場を用いない器官のバイオファブリケーションの将来への潜在的な影響をもたらす。

<table><tbody><tr><td>Geltrex</td><td>Invitrogen&amp;nbsp;</td><td>A1413202</td><td></td></tr><tr><td>Trypsin/EDTA 0.05%</td><td>Thermo Fisher</td><td>15400054</td><td></td></tr><tr><td>Defined Trypsin inhibitor 0.0125%</td><td>Thermo Fisher</td><td>R007100</td><td></td></tr><tr><td>RPMI Cell Media</td><td>Invitrogen</td><td>11875-093</td><td>RPMI supplemented with B27 constitutes HIPSC-CM culture media</td></tr><tr><td>B-27 Supplement</td><td>Thermo Fisher</td><td>17504044</td><td>RPMI supplemented with B27 constitutes HIPSC-CM culture media</td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Human cardiac fibroblasts (adult ventricular type)</td><td>Sciencell</td><td>6310</td><td></td></tr><tr><td>Human umbilical vein endothelial cells</td><td>Lonza</td><td>CC-2935</td><td></td></tr><tr><td>PrimeSurface ultra-low attachment 96-well U-bottom plates&amp;nbsp;</td><td>Akita Sumitomo Bakelite Co.</td><td>MS-9096UZ</td><td></td></tr><tr><td>Regenova Bio 3D Printer</td><td>Cyfuse Biomedical K.K.</td><td>N/A</td><td>www.cyfusebio.com/en/</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Troponin T Antibody</td><td>Thermo Fisher</td><td>701620</td><td></td></tr><tr><td>Connexin 43 (Cx43) Antibody</td><td>Chemicon</td><td>MAB3068</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Thermo Fisher</td><td>P36935</td><td></td></tr></tbody></table>

creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting

このプロトコルは、細胞のみを使用して、生体材料を使用せずに心臓組織の3Dバイオプリントを記述する。心筋細胞、内皮細胞および線維芽細胞を最初に単離し、計数し、所望の細胞比で混合する。それらは、超低付着96ウェルプレート中の個々のウェルで共培養される。 3日以内にスフェロイドが鼓動する。これらの回転楕円体は、真空吸引を使用してノズルによってピックアップされ、3Dバイオプリンタを使用してニードルアレイ上に組み立てられる。スフェロイドは、次に、針アレイ上で融合することが可能となる。 3次元バイオプリンティングの3日後、スフェロイドは、すでに自発的に鼓動している無傷のパッチとして除去される。 3Dバイオプリントされた心臓パッチは、構成要素スフェロイドの機械的統合を示し、心臓組織の再生および心臓病の3Dモデルとして非常に有望である。

ステップ4.4（共培養）の終わりに、各ウェルの細胞は、超低付着96ウェルU底プレートの底に凝集して、重力によってスフェロイドを形成するはずである。これらのスフェロイドにはhiPSC-CM、HCFs、HUVECが含まれており、光学顕微鏡下で視覚的に検査することができ、2次元投影で円形に見えるはずです（ 図1 ）。ステップ6.3の終わりに、3Dバイオプ?...

Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

このプロトコルは、生体材料を使用せずに心臓組織の3Dバイオプリントを記述する。 3Dバイオプリントされた心臓パッチは、構成要素スフェロイドの機械的統合を示し、心臓組織の再生および心臓病の3Dモデルとして非常に有望である。

3D Bioprintingを用いたスフェロイドの機械的統合を示す心臓組織の創出

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

creation-cardiac-tissue-exhibiting-mechanical-integration-spheroids

Research

JoVE Journal

Bioengineering

9.9K ビュー.  Johns Hopkins Hospital. このプロトコルは、生体材料を使用せずに心臓組織の3Dバイオプリントを記述する。 3Dバイオプリントされた心臓パッチは、構成要素スフェロイドの機械的統合を示し、心臓組織の再生および心臓病の3Dモデルとして非常に有望である。 

ビデオ: Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

マイクロコンタクトプリントスルーアライン機能心筋組織の生成

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

生物工学

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

心臓細胞療法のメカニズムを研究するために定義されたヒューマン・エンジニア心臓組織の構築

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

マイクロ流体工学バイオプリンティング血管組織や Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

スキャフォールド フリー三次元心筋組織構築純金型ベース方式

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心筋梗塞のマウスモデルにおける3次元バイオプリントパッチの移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

ヒトiPSC由来心筋細胞、心臓線維芽細胞、内皮細胞を用いた3次元心臓ミクロ組織アレイの作製

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

ヒト心臓病態生理学を研究するインビトロバイオエンジニアリング心臓組織としての心臓スフェロイド

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

マイクロ流体プラットフォーム内での3D組織化ヒト心臓組織の開発

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

手術前計画と実習訓練における応用を有する患者特異的シリコーン心臓モデルの作成

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

人工心臓組織のデータ取得とモデルスループットを向上させるバイオリアクターの設計

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

3D Bioprintingを用いたスフェロイドの機械的統合を示す心臓組織の創出

要約

さらに動画を探す

この動画の章

マイクロコンタクトプリントスルーアライン機能心筋組織の生成

心臓細胞療法のメカニズムを研究するために定義されたヒューマン・エンジニア心臓組織の構築

マイクロ流体工学バイオプリンティング血管組織や Organoids

スキャフォールドフリー三次元心筋組織構築純金型ベース方式

心筋梗塞のマウスモデルにおける3次元バイオプリントパッチの移植

ヒトiPSC由来心筋細胞、心臓線維芽細胞、内皮細胞を用いた3次元心臓ミクロ組織アレイの作製

ヒト心臓病態生理学を研究するインビトロバイオエンジニアリング心臓組織としての心臓スフェロイド

マイクロ流体プラットフォーム内での3D組織化ヒト心臓組織の開発

手術前計画と実習訓練における応用を有する患者特異的シリコーン心臓モデルの作成

人工心臓組織のデータ取得とモデルスループットを向上させるバイオリアクターの設計

3D Bioprintingを用いたスフェロイドの機械的統合を示す心臓組織の創出

要約

さらに動画を探す

この動画の章

マイクロコンタクトプリントスルーアライン機能心筋組織の生成

心臓細胞療法のメカニズムを研究するために定義されたヒューマン・エンジニア心臓組織の構築

マイクロ流体工学バイオプリンティング血管組織や Organoids

スキャフォールド フリー三次元心筋組織構築純金型ベース方式

心筋梗塞のマウスモデルにおける3次元バイオプリントパッチの移植

ヒトiPSC由来心筋細胞、心臓線維芽細胞、内皮細胞を用いた3次元心臓ミクロ組織アレイの作製

ヒト心臓病態生理学を研究するインビトロバイオエンジニアリング心臓組織としての心臓スフェロイド

マイクロ流体プラットフォーム内での3D組織化ヒト心臓組織の開発

手術前計画と実習訓練における応用を有する患者特異的シリコーン心臓モデルの作成

人工心臓組織のデータ取得とモデルスループットを向上させるバイオリアクターの設計

スキャフォールドフリー三次元心筋組織構築純金型ベース方式