Name: construction of defined human engineered cardiac tissues to study mechanisms of cardiac cell therapy
Uploaded: 2016-03-01T13:00:00
Description: Watch this Scientific Journal Video about Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy at JoVE.com

この作品は、BDG、アメリカに香港TRS T13-706 / 11（KDC）、NIH（R01 HL113499）の研究助成協議会、KDCにTJC、NIH / NHLBI PEN契約HHSN268201000045CにNIH（1F30HL118923-01A1）によってサポートされていましたRJ、および香港特別行政区の研究助成評議会（TBRS、T13-706 / 11）に心臓協会（12PRE12060254）追加の資金調達をRLには、NIH DRB 5T32GM008553-18によって、およびシステムと発達におけるNIDCR、学際的訓練に訓練生としてTJCに提供されました生物学と先天異常T32HD075735。また、作者は感謝して技術支援のためのバイオリアクターとMamdouh Eldalyの加工の支援のためにニューヨークのシティカレッジのザーンセンターでアーサーAutzを認識したいです。我々はまた、寛大にヒト間葉系幹細胞を提供するための心臓分化に関するアドバイス博士ケネスBoheler、博士ジョシュアウサギに感謝します。

注：HEPAろ過クラスII生物学的安全キャビネットを使用して、無菌状態のすべてのセルの操作を実行し、0.2μmのフィルターを介してそれらをフィルタリングすることにより、すべてのソリューションを殺菌。同じ無菌状態または層流フードのいずれかで組織の構築と機能のテストを実行します。 心臓分化のための準備でH7のhESCの1播種<ol><li> （1日目）基底膜マトリックス?...

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Rev. 12 (3-4), 331-343 (2007).</li><li>Taegtmeyer, H., Sen, S., Vela, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Return+to+the+fetal+gene+program:+a+suggested+metabolic+link+to+gene+expression+in+the+heart.">Return to the fetal gene program: a suggested metabolic link to gene expression in the heart.</a> Ann. N. Y. Acad. Sci. 1188, 191-198 (2010).</li></ol>

定義された人間工学的心臓組織（HECT）の構築は、人間の心筋細胞機能のより一貫性と信頼性の高いモデルを提供することができます。批判的に、システム内のすべての細胞および細胞外成分が知られており、従って、分化過程に起因する他の未知の細胞型の交絡の影響を除去し、所望のように操作することができます。急速な細胞増殖と高い収率のバランスをとるためには、分化は、理想的...

心臓組織工学は、より最近になって、両方のマウス心筋1-6と、から作られた完全に機能する、鼓動組織、ヒト幹細胞由来の心筋細胞7-12の結果を公表する複数のグループで、10年間で大きく前進しました。心臓組織工学の分野は、二つの主要な本質的に独立した目標によって駆動される：1）機能4-6を改善することができない心臓に移植することができる外因性の移植片を開発します。及び2）生理学および疾患を研究するためのin vitroモデルの開発、または治療開発の2,7のためのスクリーニングツールとしてします。 三次元（3-D）細胞培養物を、3-Dマトリックスは、従来の2次元単層細胞培養物よりも自然な心臓の微小環境を反映するように、次世代のスクリーニングツールを開発するために必須であると考えられます。実際に細胞生物学のいくつかの側面は、2次元対3次元培養13,14根本的に異なっています</suP&gt;。細胞外マトリックス、および細胞集団：さらに、操作心臓組織は完全に定義された成分から構成されています。細胞外マトリックス組成物（通常のフィブリン9またはコラーゲン7,8,10）を厳密に制御しつつ、従来の操作されたヒト心臓組織のために、入力細胞組成物は、あまりの指向心臓分化の細胞の混合物全体で、定義されますか胚性幹細胞（ESC 7,9）や人工多能性幹細胞（IPSC 10,12）を組織に添加されます。特定の細胞株および使用する分化プロトコルの効率に応じて、心筋の得率（ すなわち 、ventricular-、atrial-、またはペースメーカーのような）、25％未満から90％以上に、特定の心筋細胞表現型の範囲とすることができますまたしても、非心筋細胞画分は15,16非常に不均一であることで差別心臓メートルの成熟度を変更することができ、変化させることができます17を yocytes。 最近の心臓組織工学作業は心臓レポーターヒト胚性幹細胞株8または細胞表面マーカー18は、分化の心筋細胞成分を単離するために使用されるのいずれかで、細胞のインプット集団を制御しようとしています。唯一の心筋細胞で構成最初に組織が理想的であるように思われるが、これは実際にはそうではありません。心筋細胞の1：1の比率：線維芽細胞が最も高い単収縮力8を生成するのみ心筋細胞で構成hECTsは、いくつかのグループは、3を見つけることで、機能的な組織へと圧縮することができません。生細胞の選別のための表面マーカーを含む、様々な細胞の選択方法を用いることにより、定義された細胞集団でhECTsを作成することが可能です。非心臓マーカー間質細胞は、推定上の線維芽細胞マーカーCD90 19,20のように、しばらくの間利用可能であったが、心筋細胞の表面マーカーは、より困難でした特定する。 SIRPαは、人間の心筋細胞18のために同定された最初の心臓表面マーカー間にあったと心臓系統に高度に選択的であることが示されています。最近、我々は、SIRPα+およびCD90のための二重ソートことを発見した-細胞、線維芽細胞様の表現型（Josowitz、未発表の観察）を示すCD90 +集団と、ほぼ純粋な心筋細胞が得られます。心筋細胞およびCD90 +線維芽細胞- SIRPα+ / CD90の1組み合わせ：これらの収集した知見に基づき、本明細書中で我々は3を使用してhECTsを作成する方法を説明します。 完全に定義された人間の心臓組織を設計する能力は、堅牢なスクリーニングツールを作成するためだけでなく、新興の細胞および遺伝子に基づく心臓治療を研究するためのモデルシステムを開発するだけでなく、必要不可欠です。間葉系幹細胞（MSC）を含む細胞タイプを利用心不全、特に、多数の細胞療法、21 </SUP&gt;、心臓幹細胞22及び骨髄単核細胞を23~25は 、臨床試験で試験されています。最初の結果の多くは21,23,25を約 ​​束してきたが、最初の利点は、多くの場合、時間26-29とともに減少します。同様の傾向が原因MSC補充に重要な機能的利益を表示するマウス操作心臓組織において報告されているが、利点は、長期培養1中持続しません。サブ最適な性能を根底にある細胞治療を支配するメカニズムの限られた知識です。どのように治療用細胞それらの有益な影響だけでなく、筋細胞nonmyocyte相互作用の潜在的な負の影響を及ぼし、心不全の患者のために、最小限の副作用で、臨床的に有意かつ持続的な利益をもたらす改善された治療法の開発を可能にする。より深く理解ここでは、interrogに定義されたhECTsの使用を記載しています細胞ベースの治療のメカニズムを食べました。制御された組織組成物は、心筋細胞の性能に影響を与える特定の因子を同定することが不可欠です。我々はラットECTS 1で実証したように、直接目的の治療用細胞型（ 例えば 、MSCS）でhECTsを補完する、心筋細胞のパフォーマンスへの影響を明らかにすることができます。 次の多段階プロトコルは、マルチ組織バイオリアクターの製造に続いて、組織構造および機能分析の説明を結論、指向心臓幹細胞の分化から始まります。我々の実験は、NIH-承認H7ヒト胚性幹細胞（hESCの）ラインを使用して実行されます。しかし、次のプロトコルは、追加のhESCラインと同様の結果で三誘導多能性幹細胞（hiPSC）株を用いて試験されました。我々は、HECT製造における心筋細胞分化および成功効率は、特にfoは、細胞株に依存することができることを見出しました個々の患者由来のR hiPSCライン。このプロトコルに従うことにより、2個の6ウェルディッシュは、20日間分化さと十分な6定義された組織を作るために、ソートした後、約250万筋細胞を生み出す168万のhESC（ウェル当たり14万細胞）、の合計でメッキされています。

<table border="0">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2411</td>
			<td>Prepare a 2.5 mg/ml stock in DMSO and filter-sterilize</td>
		</tr>
		<tr>
			<td>B27 with Insulin</td>
			<td>Life Technologies</td>
			<td>17505055</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 without Insulin</td>
			<td>Life Technologies</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>Create 6 &#956;M stock, then aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Essential 8 Media</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td></td>
		</tr>
		<tr>
			<td>H7 Human Embryonic Stem Cells</td>
			<td>WiCell</td>
			<td>WA07</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC Qualified Matrix, Corning Matrigel</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Thaw on ice at 4 &#176;C overnight then aliquot 150 &#956;l into separate tubes and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>IWR-1</td>
			<td>Sigma-Aldrich</td>
			<td>I0161</td>
			<td>Create 10 mM stock and aliquot. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neonatal Calf Serum</td>
			<td>Life Technologies</td>
			<td>16010159</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-enzymatic Dissociation Reagent: Gentle Cell Dissociation Reagent</td>
			<td>Stem Cell Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Keep refrigerated</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK Inhibitor)</td>
			<td>Stemgent</td>
			<td>04-0012</td>
			<td>Resuspend to a 10 mM stock concentration, aliquot and store at -20 &#176;C.&#160; Avoid freeze thaw cycles.</td>
		</tr>
		<tr>
			<td>Cell Sorting</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90-FITC</td>
			<td>BioLegend</td>
			<td>328107</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic Dissociation Reagent: Cell Detach Kit I (0.04 % Trypsin/ 0.03% EDTA, Trypsin neutralization solution and Hanks Buffered Salt Solution)&#160;</td>
			<td>PromoCell</td>
			<td>C-41200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologics</td>
			<td>S11250</td>
			<td></td>
		</tr>
		<tr>
			<td>SIRP&#945;-PE/Cy7</td>
			<td>BioLegend</td>
			<td>323807</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Construction</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/0.1% EDTA</td>
			<td>Fisher Scientific</td>
			<td>25-053-CI</td>
			<td>Optional: For collection of supplemental cells of interest</td>
		</tr>
		<tr>
			<td>10x MEM</td>
			<td>Sigma-Aldrich</td>
			<td>M0275-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS Packets</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Bovine Type I</td>
			<td>Life Technologies</td>
			<td>A10644-01</td>
			<td>Keep on ice</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagles Medium (DMEM), High Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3784</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>NC0536757</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Polystyrene Round-Bottom Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td>With integrated 35 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>50 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well flat bottom tissue-culture treated plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper, Disposable</td>
			<td>Biologix</td>
			<td>70-2180</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysulfone</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (Teflon)</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZ-61</td>
			<td>Or similar, must have a mount for the high speed camera to attach</td>
		</tr>
		<tr>
			<td>Electrical Pacing System</td>
			<td>Astro-Med, Inc</td>
			<td>Grass S88X Stimulator</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>Pixelink</td>
			<td>PL-B741U</td>
			<td>Or similar, but must be capable of 100 frames per second for accurate data acquisition</td>
		</tr>
		<tr>
			<td>Plate Temperature Control</td>
			<td></td>
			<td></td>
			<td>Used to maintain media temperature during data acqusition.</td>
		</tr>
		<tr>
			<td>Custom Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>LabView Post-tracking Program</td>
			<td></td>
			<td></td>
			<td>available upon request from the authors</td>
		</tr>
	</tbody>
</table>

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.

心筋細胞を得るためには、Bohelerとリアン分化方法を少し変更したバージョンは30,31に使用されます。分化は、細胞増殖の対数期の間に開始することなく、出発集団（約75％が最適である）選別後の細胞の使用可能な数を得るために十分にコンフルエントであることが肝要です。典型的に、H7ヒトES細胞のために、37℃で維持不可欠8培地、5％CO 2インキュベーター中で6ウェル皿の...

Watch this Scientific Journal Video about Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy at JoVE.com

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

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 ...

Research

JoVE Journal

Bioengineering

Shock Wave Application to Cell Cultures

細胞培養物衝撃波アプリケーション

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

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

ミニチュア組織培養系でマウス心臓弁の培養

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

心臓医薬品安全性スクリーニングのための人間エンジニア心臓組織の自動収縮分析

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

ひと血清における心筋バイオ マーカーを定量化する臨時の光伝送を使用してください。

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

成人骨髄由来造血幹細胞が細胞工学磁気ターゲッティングと結合を有効にするにマイクロ Rna の転送のためのプロトコル

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

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

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

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

ヒトの神経ノチカルスライスの二重電圧およびカルシウム光学マッピングによる前臨床心臓電気生理学的評価

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

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

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

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

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 ...