Name: a net mold-based method of scaffold-free three-dimensional cardiac tissue creation
Uploaded: 2018-08-05T13:00:00
Description: Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

著者は、次の資金源を認める: 心血管の研究のため魔法、事項の資金。

1. 心筋細胞の調製
<ol><li>17と前述したように、コート 6 ウェル基底膜マトリックスと文化人間誘導された多能性幹細胞 (hiPSCs)。</li>
	<li>上記の方法18を使用してによる CMs に hiPSCs を区別します。</li>
	<li>16-18 d 後の分化には、カルシウムやマグネシウム、孵化 1 ml/トリプシンまたは細胞解離試薬 (のテーブルを参照のも続いてせず、リン酸緩衝生理食?...

<ol>
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L., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Microscale+Devices+to+3D+Printing:+Advances+in+Fabrication+of+3D+Cardiovascular+Tissues.">From Microscale Devices to 3D Printing: Advances in Fabrication of 3D Cardiovascular Tissues.</a> Circulation Research. 120 (1), 150-165 (2017).</li><li>Tandon, N., 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=Electrical+stimulation+systems+for+cardiac+tissue+engineering.">Electrical stimulation systems for cardiac tissue engineering.</a> Nature Protocols. 4 (2), 155-173 (2009).</li><li>Duran, A. G., 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=Regenerative+Medicine/Cardiac+Cell+Therapy:+Pluripotent+Stem+Cells.">Regenerative Medicine/Cardiac Cell Therapy: Pluripotent Stem Cells.</a> The Journal of Thoracic and Cardiovascular Surgery. 66 (1), 53-62 (2018).</li><li>Lux, 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=In+vitro+maturation+of+large-scale+cardiac+patches+based+on+a+perfusable+starter+matrix+by+cyclic+mechanical+stimulation.">In vitro maturation of large-scale cardiac patches based on a perfusable starter matrix by cyclic mechanical stimulation.</a> Acta Biomaterialia. 30, 177-187 (2016).</li><li>Eschenhagen, 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=Three-dimensional+reconstitution+of+embryonic+cardiomyocytes+in+a+collagen+matrix:+a+new+heart+muscle+model+system.">Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system.</a> The FASEB Journal. 11 (8), 683-694 (1997).</li><li>Moldovan, N. 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K., Shinoka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+cardiac+patch+seeded+with+human+induced+pluripotent+stem+cell+derived+cardiomyocytes+promoted+the+regeneration+of+host+cardiomyocytes+in+a+rat+model.">Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model.</a> Journal of Cardiothoracic Surgery. 11 (1), 163 (2016).</li><li>Caspi, O., 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=Tissue+engineering+of+vascularized+cardiac+muscle+from+human+embryonic+stem+cells.">Tissue engineering of vascularized cardiac muscle from human embryonic stem cells.</a> Circulation Research. 100 (2), 263-272 (2007).</li><li>Ong, C. S., 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=Biomaterial-Free+Three-Dimensional+Bioprinting+of+Cardiac+Tissue+using+Human+Induced+Pluripotent+Stem+Cell+Derived+Cardiomyocytes.">Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes.</a> Scientific Reports. 7 (1), 4566 (2017).</li><li>Kelm, J. 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=A+novel+concept+for+scaffold-free+vessel+tissue+engineering:+self-assembly+of+microtissue+building+blocks.">A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks.</a> Journal of Biotechnology. 148 (1), 46-55 (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> The Journal of Heart and Lung Transplantation. 35 (1), 137-145 (2016).</li><li>Zuppinger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+culture+for+cardiac+cells.">3D culture for cardiac cells.</a> Biochimica et Biophysica Acta. 1863 (7 Pt B), 1873-1881 (2016).</li><li>Langenbach, F., 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=Generation+and+differentiation+of+microtissues+from+multipotent+precursor+cells+for+use+in+tissue+engineering.">Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering.</a> Nature Protocols. 6 (11), 1726-1735 (2011).</li><li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends in Biotechnology. 31 (2), 108-115 (2013).</li><li>Agocha, A., Sigel, A. V., Eghbali-Webb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adult+human+heart+fibroblasts+in+culture:+a+comparative+study+of+growth,+proliferation+and+collagen+production+in+human+and+rabbit+cardiac+fibroblasts+and+their+response+to+transforming+growth+factor-beta1.">Characterization of adult human heart fibroblasts in culture: a comparative study of growth, proliferation and collagen production in human and rabbit cardiac fibroblasts and their response to transforming growth factor-beta1.</a> Cell Tissue Research. 288 (1), 87-93 (1997).</li><li>Baudin, B., Bruneel, A., Bosselut, N., Vaubourdolle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+isolation+and+culture+of+human+umbilical+vein+endothelial+cells.">A protocol for isolation and culture of human umbilical vein endothelial cells.</a> Nature Protocols. 2 (3), 481-485 (2007).</li><li>Pimentel, C. 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=Three-dimensional+fabrication+of+thick+and+densely+populated+soft+constructs+with+complex+and+actively+perfused+channel+network.">Three-dimensional fabrication of thick and densely populated soft constructs with complex and actively perfused channel network.</a> Acta Biomaterialia. 65, 174-184 (2018).</li><li>Shimizu, 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=Polysurgery+of+cell+sheet+grafts+overcomes+diffusion+limits+to+produce+thick,+vascularized+myocardial+tissues.">Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues.</a> The FASEB Journal. 20 (6), 708-710 (2006).</li><li>Sakaguchi, K., Shimizu, T., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+three-dimensional+vascularized+cardiac+tissue+with+cell+sheet+engineering.">Construction of three-dimensional vascularized cardiac tissue with cell sheet engineering.</a> Journal of Controlled Release. 205, 83-88 (2015).</li><li>Ong, C. S., 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=Creation+of+Cardiac+Tissue+Exhibiting+Mechanical+Integration+of+Spheroids+Using+3D+Bioprinting.">Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting.</a> Journal of Visualized Experiments. 125 (e55438), (2017).</li><li>Tan, 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=Cell+number+per+spheroid+and+electrical+conductivity+of+nanowires+influence+the+function+of+silicon+nanowired+human+cardiac+spheroids.">Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids.</a> Acta Biomaterialia. 51, 495-504 (2017).</li><li>Karra, R., Poss, 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=Redirecting+cardiac+growth+mechanisms+for+therapeutic+regeneration.">Redirecting cardiac growth mechanisms for therapeutic regeneration.</a> Journal of Clinical Investigation. 127 (2), 427-436 (2017).</li><li>Bartholoma, P., 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=Three-dimensional+in+vitro+reaggregates+of+embryonic+cardiomyocytes:+a+potential+model+system+for+monitoring+effects+of+bioactive+agents.">Three-dimensional in vitro reaggregates of embryonic cardiomyocytes: a potential model system for monitoring effects of bioactive agents.</a> Journal of Biomolecular Screening. 10 (8), 814-822 (2005).</li></ol>

この法の意義は、その再現性と結果の多層の心臓組織の有効性であります。心臓組織工学分野での現在の目標の一つ、打撃、多層、および機能の 3次元心筋パッチを作成する方法を特定します。心筋細胞、血管内皮細胞、線維芽細胞から成る小説ネット型に回転楕円体の手動直播による多層心臓組織を作成する効率的かつ再現性のある方法を報告する.このメソッドで使用される純金型は、様?...

現在の心臓組織工学の目的は、交換または構造と負傷した心筋組織1の機能を修復する治療法を開発することです。ネイティブの心臓組織の重要な収縮と電気生理学的特性を示す心筋組織の 3次元モデルを作成する方法には2,3を急速に拡大しています。様々 な戦略を模索、研究4,5で使用されます。これらの方法はゼラチン、コラーゲン、フィブリン、ペプチド6などの合成と天然特定の生理活性物質ゲルの使用からバイオ インク蒸着の技術2バイオプリンティング技術の7まであります。
足場自由な方法が外部足場材料8を組み込むことの欠点がなく、生体材料ベースのメソッドとして匹敵する組織を作り出すことができることが示されています。オレン カスピーらは、種々 の細胞の結合が高度に血管人間設計された心臓ティッシュ9の生成を有効を示した。あごらは、回転楕円体から心臓のパッチを作成するための 3次元印刷法を開発しました。結果として得られるパッチ参照比10で血管内皮細胞、線維芽細胞、心筋細胞で構成されます。回転楕円体は、効果的な「ビルディング ブロック」足場無料心臓組織の作成の彼らの低酸素状態に対する耐性があるし、注入11,12の十分な機械的完全性を持って示されています。以前の研究は、回転楕円体を作成するためのいくつかの製造方法を示している、絞首刑の使用を含むドロップ メソッド、スピナー フラスコ13、マイクロ流体システム14、および非付着性文化の表面光沢をめっきしたものアガロース マイクロ金型15。このプロトコルでは絞首刑を使用してドロップ デバイスは、一度に何百もの回転楕円体を凝縮の細孔が含まれています。
本研究は、小説と正方形の金型に、回転楕円体を手動でシードとシェーカーで組織の成熟をインキュベートを含む心臓組織の創造のための効率的な足場無料メソッドを示します。いつも静置培養条件下で酸素の拡散は中心壊死をもたらす組織構成要素の外側の側面に限定されます。ただし、ネット型で金型にシードすべての楕円は、栄養素や酸素の増加の拡散を可能にする一定の流体運動とメディアに没頭しています。また、この金型ベースのメソッドは、最小限の労力でサイズの異なる組織パッチの同時作成、金型から得られた組織を簡単に削除できます。この手法スキャフォールド フリー、多層の心筋パッチの効率的かつ再現可能な作成できます。

<table border="0" cellpadding="0" cellspacing="0" style="width:708px;" width="708">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Human Cardiac fibroblasts (HCF)</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">6310</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">FM-2 Consists of Basal Medium</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">2331</td>
			<td style="width:220px;">HCF culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Human umbilical vein endothelial cells (HUVEC)</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EGM+Bullet Kit&#160;</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC5035</td>
			<td>HUVEC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">E8 media&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1517001</td>
			<td>HiPSC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Geltrex&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1413202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">TrypLE Express Enzyme (1X)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">12604013</td>
			<td>Trypsin and Cell dissociation reagent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">RPMI media</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">11875093</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">B-27 supplement (50x)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">17504044</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Novel net mold&#160;</td>
			<td style="width:139px;">TissueByNet Co.,Ltd</td>
			<td style="width:119px;">NM25-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hanging drop plate</td>
			<td style="width:139px;">Kuraray Co.,Ltd</td>
			<td style="width:119px;">MPc350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">6 well plates&#160;</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS-3516</td>
			<td></td>
		</tr>
	</tbody>
</table>

a net mold-based method of scaffold-free three-dimensional cardiac tissue creation

このプロトコルでは、小説と追加足場材料なし三次元 (3 D) の心筋組織を作成する簡単な純金型ベース方法をについて説明します。人間誘導された多能性幹細胞由来心筋細胞 (iPSC CMs)、心臓 (HCFs)、線維芽細胞およびひと臍帯静脈血管内皮細胞 (Huvec) は、隔離され、70 %ipsc - cms、15 %hcfs と 15 %huvec 細胞懸濁液を生成するために使用します。回転楕円体の数百人を一度に凝縮用孔を含む超低添付ファイル「ハンギング ドロップ」システムの共培養しています。セルを集計、自発的に共培養 3 日後暴行スフェロイドを形成します。スフェロイドは収穫され、新規金型に播種、シェーカーのインキュベーター内で培養しました。スフェロイドは播種後約 7 日成熟した機能的な組織になります。結果多層組織は満足のいく構造的整合性と同期打撃動作溶かされた回転楕円体から成っています。この新しいメソッドでは、将来的に心不全の治療のために設計された組織を作成する再現性とコスト効果の高い方法として有望な可能性があります。

実験では、我々 は 70% iPSC CMs、15% HCFs mL あたり 2,475,000 細胞の濃度で RPMI/B-27 携帯メディアで 15 %huvec の細胞懸濁液を利用しました。プロトコルの手順 4.3 細胞懸濁液を作成した後は、ハンギング ドロップ システム、超低添付ファイルの各ウェルに細胞懸濁液 4 mL を分配しました。絞首刑の使用ドロップ システムは湿度 95%、5% CO2、37 ° C で培養 3 日後回転楕円?...

Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

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

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

このプロトコルでは、満足のいく構造的整合性と同期打撃動作立体足場無料心臓組織を作成する純金型ベースの方法について説明します。

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

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

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

三次元組織配置に発達メカニズムを要約し、神経系の再生を容易にするアストロ サイト ネットワーク

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Melt Electrospinning Writing of Three-dimensional PolyÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Âµ-caprolactone Scaffolds with Controllable Morphologies for Tissue Engineering Applications

組織工学的応用の制御可能な形態の三次元 Poly(ε-caprolactone) 足場のエレクトロスピニング記述を溶かす

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

三次元ペプチド足場で哺乳動物の細胞を培養

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

腎組織のプライマリ セグメントと新規スキャフォールド フリー構造技術と定款の

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

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

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