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、リン酸緩衝生理食塩水 (PBS) x 1 の 2 mL の各ウェルを洗浄によって心筋細胞を一時中断します。材料) 室温で 5 分間。</li>
	<li>トリプシンまたは細胞解離試薬を中和 (材料表参照) ロズウェル パーク記念研究所 (RPMI) セル培地 B-27 (RPMI/B-27 携帯メディア) の等量を使用して。上下のピペット、付着細胞を緩める。</li>
	<li>10 mL の血清ピペットで中断された心筋細胞を収集し、50 mL の円錐管にそれらを転送します。</li>
	<li>250 x g細胞ペレットを取得するために室温で 5 分間で細胞懸濁液を遠心します。</li>
	<li>RPMI/B-27 携帯メディアの 10 mL にペレットを再懸濁します。</li>
	<li>細胞懸濁液の 20 μ L を組み合わせて 0.4% トリパン ブルー溶液の同量入れ、軽く混ぜます。</li>
	<li>マニュアルの検定を使用してカウントし、細胞懸濁液の濃度と細胞の生存率を入手します。</li>
</ol>2. 線維芽細胞の作製
<ol><li>16を前述のよう人間心臓線維芽細胞 (HCF) (成人心室型) 細胞株の培養を開始します。</li>
	<li>トリプシンまたはセルの解離試薬の適切な量でそれらをインキュベートし、HCFs を中断 (材料表参照) 室温16で 5 分間。T175 フラスコ 10 mL のトリプシンまたはセルの解離試薬の使用しています。</li>
	<li>トリプシンまたは細胞解離試薬を中和 (材料の表を参照してください)、50 mL の円錐管にサンプルを転送し、250 x gを入手する部屋の温度で 5 分間で細胞懸濁液を遠心分離媒体の等量を使用して、ペレット。</li>
	<li>線維芽細胞成長培地 10 mL にペレットを再懸濁します (材料の表を参照してください)。</li>
	<li>HCF 細胞懸濁液の 20 μ L を組み合わせて 0.4% トリパン ブルー溶液の同量入れ、軽く混ぜます。</li>
	<li>自動化された細胞カウンターまたは手動検定を使用してカウントし、新しい細胞懸濁液の濃度と細胞の生存率を入手します。</li>
</ol>3. 血管内皮細胞の調製
<ol><li>ひと臍帯静脈内皮細胞 (株) 線、17を前述の培養を開始します。トリプシンまたはセルの解離試薬の適切な量でそれらをインキュベートし、比較を中断 (材料表参照) 室温17で 3 分間。トリプシンまたは細胞解離試薬を中和する (材料の表を参照してください)、50 mL の円錐管に転送中の等量を使用して、250 x gペレットを取得するために室温で 5 分間で細胞懸濁液を遠心分離します。 注: T175 フラスコ 10 mL のトリプシンまたはセルの解離試薬が使用されました。</li>
	<li>内皮細胞成長培地 10 mL にペレットを再懸濁します (材料の表を参照してください)。</li>
	<li>新生活懸濁液 20 μ L を組み合わせると 0.4% トリパン ブルー溶液の同量とそれを汚す、軽く混ぜます。</li>
	<li>自動化された細胞カウンターまたは手動検定を使用してカウントし、細胞懸濁液の濃度と細胞の生存率を入手します。</li>
</ol>4. ドロップ回転楕円体の作成
<ol><li>絞首刑の場所ドロップ デバイス滅菌 6 ウェル プレートに 850 の細孔 (それぞれ 350 μ m の直径) が含まれています。</li>
	<li>前述のようにセルの 3 種類を分離: による CMs、HCFs、および比較します。1 mL あたり 2,475,000 細胞の濃度で RPMI/B-27 携帯メディアで 70% iPSC CMs、15 %hcfs と 50 mL の円錐管に 15 %huvec の比率で結合します。</li>
	<li>ハンギング 6 ウェル プレートに装着されているドロップ システム (350 μ m の細孔径) と超低添付ファイルの各ウェルに 4 mL (1 mL あたり 2,475,000 細胞) の細胞懸濁液を調剤します。</li>
	<li>回転楕円体が絞首刑に細胞懸濁液を調剤の 12 h の中で形成される自発的にドロップ デバイス。37 ° C、5% CO2、湿度 95%、72 時間 (3 d) の合計のための文化、回転楕円体の成熟のために続けます。</li>
	<li>文化の 72 時間後収穫は絞首刑の下部孔を通って回転楕円体中皿にデバイスを配置し、絞首刑からスフェロイドをリリースに優しくそれを旋回システムをドロップ ドロップ デバイス。</li>
</ol>5. 3-D パッチ作成新規純金型を使用
<ol><li>基本、底正方形板、底網、10 レイヤード サイド滅菌ピンセットのペアの助けを借りて新規金型を作成する網をまとめます。まず、スタック ベースで底正方形板を合わせ、コーナー ポストを使用してネットを底します。スタックし、層の良いステンレス製熊手の網を作成する方向を交互に 10 側網。</li>
	<li>表面張力を軽減し、その後、充填ベースの上に組み立てられた金型を転送 PBS またはメディア ベースの充填をフラッシュします。</li>
	<li>PBS と同じ方法でメディア ネット金型キャビティを濡れています。希望するサイズの前提の空洞に、回転楕円体をゆっくり記入 (2 × 2 × 1 mm、4 × 4 × 1 mm、または 6 × 6 × 1 mm)。 注: ので、細胞凝集塊はタイトな接続で、静的なまま、空洞に回転楕円体の予想量の 120% を供給することを確認します。</li>
	<li>Net トップ、コーナー ポストの上に四角い天板を組み立てる、ストッパーとスフェロイドのにじみを防ぐためにホールディング チューブを固定します。</li>
	<li>いっぱいネット金型システムを 6 ml RPMI/B-27 携帯メディアの全体の組み立てられた金型をカバーする十分な培地で無菌容器に 6 ウェル プレートに転送します。</li>
	<li>3,000-4,000 x gで 7-10 日間スイング シェーカーで塗りつぶされた金型システム 6 ウェル プレートを孵化させなさい。 注: 孵化の持続期間は、心臓のパッチのサイズによって決定されます。大規模なパッチ、インキュベーション時間心臓パッチ成熟増加すべき必要があります。</li>
</ol>6. 新規純金型からのパッチの削除
<ol><li>メディアから金型システムを取り外して滅菌 10 cm 皿に滅菌処理マットの上に置きます。</li>
	<li>ホールディング チューブ、ストッパー、正方形天板上の net を削除します。慎重に滅菌ピンセットのペアを持つ層状側網 1 つずつをスライドさせます。抜後、そのまま心臓パッチは、ネットの底の上に取得されます。</li>
	<li>滅菌鉗子を使用してパッチでネットの下をピックアップし、RPMI/B27 メディアの 5 ml 35 mm ディッシュにネットの下を転送します。軽く料理、または無菌細胞スクレーパーとネットをゆっくりと旋回してネットの下からパッチを緩めます。ネットの下から剥離後 RPMI/B27 メディアで心筋組織を培養することができます。</li>
	<li>35 mm ディッシュの浮遊 3次元心筋パッチをインキュベートし続けます。機能同期鼓動は、鋳型から取り出した後、早ければ 24 時間観察できます。</li>
	<li>Net の金型からパッチを削除した後整合性と時間同期暴行増加の強度とその形状が維持されることを確認します。 注: 3-D ネット金型ベース心筋パッチ抜後コンポーネント cardiospheres の電気的統合を出展しました。60 から 80 ビート/分 60 日間以上の永続化に至るまで、周波数を見ました。</li>
</ol>

<ol>
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著者が明らかに何もありません。

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><tbody><tr><td>Human Cardiac fibroblasts (HCF)</td><td>Sciencell</td><td>6310</td><td></td></tr><tr><td>FM-2 Consists of Basal Medium</td><td>Sciencell</td><td>2331</td><td>HCF culture medium</td></tr><tr><td>Human umbilical vein endothelial cells (HUVEC)</td><td>Lonza</td><td>CC-2935</td><td></td></tr><tr><td>EGM+Bullet Kit&amp;nbsp;</td><td>Lonza</td><td>CC5035</td><td>HUVEC culture medium</td></tr><tr><td>E8 media&amp;nbsp;</td><td>Invitrogen</td><td>A1517001</td><td>HiPSC culture medium</td></tr><tr><td>Geltrex&amp;nbsp;</td><td>Invitrogen</td><td>A1413202</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X)</td><td>Thermo Fisher</td><td>12604013</td><td>Trypsin and Cell dissociation reagent</td></tr><tr><td>RPMI media</td><td>Invitrogen</td><td>11875093</td><td>RPMI media with B-27 supplement is hiPSC-CM culture medium</td></tr><tr><td>B-27 supplement (50x)</td><td>Thermo Fisher</td><td>17504044</td><td>RPMI media with B-27 supplement is hiPSC-CM culture medium</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Novel net mold&amp;nbsp;</td><td>TissueByNet Co.,Ltd</td><td>NM25-1</td><td></td></tr><tr><td>Hanging drop plate</td><td>Kuraray Co.,Ltd</td><td>MPc350</td><td></td></tr><tr><td>6 well plates&amp;nbsp;</td><td>Sigma-Aldrich</td><td>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. ...

a-net-mold-based-method-scaffold-free-three-dimensional-cardiac

Research

JoVE Journal

Bioengineering

8.8K ビュー.  The First Hospital of Jilin University. 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。

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

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

心臓組織工学のためのキャピラリーフォースリソグラフィ

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

生物工学

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. ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

組織工学：層状細胞シートの送達のための多細胞3D足場の構築

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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 ...

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

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

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 ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

生体内心筋修復用ヒトiPS細胞由来のメッシュ状の設計心臓組織の調製

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

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 ...

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, ...

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 ...

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

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心臓組織工学のためのキャピラリーフォースリソグラフィ

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

組織工学：層状細胞シートの送達のための多細胞3D足場の構築

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

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

生体内心筋修復用ヒトiPS細胞由来のメッシュ状の設計心臓組織の調製

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

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

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

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

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心臓組織工学のためのキャピラリーフォースリソグラフィ

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

組織工学：層状細胞シートの送達のための多細胞3D足場の構築

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

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

生体内心筋修復用ヒトiPS細胞由来のメッシュ状の設計心臓組織の調製

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

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

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

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