Name: preparation of human myocardial tissue for long-term cultivation
Uploaded: 2022-06-02T14:10:00
Description: Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

研究は、DZHK助成金81Z0600207(JH、PS、およびDM)および81X2600253(ADおよびTS)によって資金提供されました。
著者らは、Claudia Fahney、Mei-Ping Wu、Matthias Semisch氏に、セットアップの準備における支援と、組織培養の定期的なメンテナンスに感謝したい。

ここに記載されている実験のための組織コレクションは、ミュンヘン大学およびルール大学ボーフムの治験審査委員会によって承認されました。研究はヘルシンキ宣言ガイドラインに従って実施された。患者は、組織採取前に書面によるインフォームドコンセントを与えた。
1. 組織取得
<ol><li>心臓移植または心臓手術を受けている患者からヒト組織を得る?...

<ol>
	<li>George, S. A., Brennan, J. A., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+cardiac+electrophysiology+assessment+by+dual+voltage+and+calcium+optical+mapping+of+human+organotypic+cardiac+slices.">Preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices.</a> Journal of Visualized Expereiments: JoVE. (160), e60781 (2020).</li><li>Lu, K., 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=Progressive+stretch+enhances+growth+and+maturation+of+3D+stem-cell-derived+myocardium.">Progressive stretch enhances growth and maturation of 3D stem-cell-derived myocardium.</a> Theranostics. 11 (13), 6138-6153 (2021).</li><li>Pontes Soares, C., 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=2D+and+3D-organized+cardiac+cells+shows+differences+in+cellular+morphology,+adhesion+junctions,+presence+of+myofibrils+and+protein+expression.">2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression.</a> PloS one. 7 (5), 38147 (2012).</li><li>Klumm, M. J., 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=Long-term+cultivation+of+human+atrial+myocardium.">Long-term cultivation of human atrial myocardium.</a> Frontiers in Physiology. 13, 839139 (2022).</li><li>Krane, 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=Sequential+defects+in+cardiac+lineage+commitment+and+maturation+cause+hypoplastic+left+heart+syndrome.">Sequential defects in cardiac lineage commitment and maturation cause hypoplastic left heart syndrome.</a> Circulation. 144 (17), 1409-1428 (2021).</li><li>Miller, 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=Heart+slice+culture+system+reliably+demonstrates+clinical+drug-related+cardiotoxicity.">Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity.</a> Toxicology and Applied Pharmacology. , 406 (2020).</li><li>Fischer, C., 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=Long-term+functional+and+structural+preservation+of+precision-cut+human+myocardium+under+continuous+electromechanical+stimulation+in+vitro.">Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.</a> Nature Communications. 10 (1), 1-12 (2019).</li><li>Kang, C., 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=Human+organotypic+cultured+cardiac+slices:+new+platform+for+high+throughput+preclinical+human+trials.">Human organotypic cultured cardiac slices: new platform for high throughput preclinical human trials.</a> Scientific Reports. 6 (1), 1-13 (2016).</li><li>Ou, Q., 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=Slicing+and+culturing+pig+hearts+under+physiological+conditions.">Slicing and culturing pig hearts under physiological conditions.</a> Journal of Visualized Experiments: JoVE. (157), e60913 (2020).</li><li>Ou, Q., 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=Physiological+biomimetic+culture+system+for+pig+and+human+heart+slices.">Physiological biomimetic culture system for pig and human heart slices.</a> Circulation research. 125 (6), 628-642 (2019).</li><li>Watson, S. A., 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=Biomimetic+electromechanical+stimulation+to+maintain+adult+myocardial+slices+in+vitro.">Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.</a> Nature Communications. 10 (1), 1-15 (2019).</li><li>Abu-Khousa, 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=The+degree+of+t-system+remodeling+predicts+negative+force-frequency+relationship+and+prolonged+relaxation+time+in+failing+human+myocardium.">The degree of t-system remodeling predicts negative force-frequency relationship and prolonged relaxation time in failing human myocardium.</a> Frontiers in Physiology. 11, 182 (2020).</li><li>Bojkova, 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=SARS-CoV-2+infects+and+induces+cytotoxic+effects+in+human+cardiomyocytes.">SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.</a> Cardiovascular Research. 116 (14), 2207-2215 (2020).</li><li>Esfandyari, D. A. -. 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=MicroRNA-365+regulates+human+cardiac+action+potential+duration.">MicroRNA-365 regulates human cardiac action potential duration.</a> Nature Communications. 13 (1), 1-15 (2022).</li><li>Moretti, A., 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=Somatic+gene+editing+ameliorates+skeletal+and+cardiac+muscle+failure+in+pig+and+human+models+of+Duchenne+muscular+dystrophy.">Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.</a> Nature Medicine. 26 (2), 207-214 (2020).</li><li>Pitoulis, F. 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=Remodelling+of+adult+cardiac+tissue+subjected+to+physiological+and+pathological+mechanical+load+in+vitro.">Remodelling of adult cardiac tissue subjected to physiological and pathological mechanical load in vitro.</a> Cardiovascular Research. 118 (3), 814-827 (2021).</li><li>Curtis, T. M., Scholfield, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nifedipine+blocks+Ca2++store+refilling+through+a+pathway+not+involving+L-type+Ca2++channels+in+rabbit+arteriolar+smooth+muscle.">Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.</a> The Journal of Physiology. 532 (3), 609-623 (2001).</li><li>de Weille, J. R., Schweitz, H., Maes, P., Tartar, A., Lazdunski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calciseptine,+a+peptide+isolated+from+black+mamba+venom,+is+a+specific+blocker+of+the+L-type+calcium+channel.">Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.</a> Proceedings of the National Academy of Sciences. 88 (6), 2437-2440 (1991).</li><li>Schleifer, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+molecular+modelling+study+of+the+calcium+channel+blockers+nifedipine+and+black+mamba+toxin+FS2.">Comparative molecular modelling study of the calcium channel blockers nifedipine and black mamba toxin FS2.</a> Journal of Computer-Aided Molecular Design. 11 (5), 491-501 (1997).</li><li>Thomas, G., Chung, M., Cohen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dihydropyridine+(Bay-K8644)+that+enhances+calcium+currents+in+guinea+pig+and+calf+myocardial+cells.+A+new+type+of+positive+inotropic+agent.">A dihydropyridine (Bay-K8644) that enhances calcium currents in guinea pig and calf myocardial cells. A new type of positive inotropic agent.</a> Circulation Research. 56 (1), 87-96 (1985).</li><li>Pitoulis, F. G., Watson, S. A., Perbellini, F., Terracciano, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+slices+come+to+age:+an+intermediate+complexity+in+vitro+cardiac+model+for+translational+research.">Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research.</a> Cardiovascular Research. 116 (7), 1275-1287 (2020).</li><li>Watson, S. A., 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=Preparation+of+viable+adult+ventricular+myocardial+slices+from+large+and+small+mammals.">Preparation of viable adult ventricular myocardial slices from large and small mammals.</a> Nature Protocols. 12 (12), 2623-2639 (2017).</li></ol>

過去には、心臓血管研究は心筋細胞の培養において大きな進歩を遂げました。しかし、無傷の幾何学的形状を有する心筋細胞の3D培養はまだ十分に確立されていない。心筋組織の エクス ビボ培養に適用された以前のプロトコルと比較して、ここで説明したプロトコルは、組織の インビボ 環境により密接に似ている。さらに、前負荷および後負荷の適用は、より生体模倣的な環?...

過去10年間で、心筋細胞のインビトロ培養は、2Dおよび三次元(3D)技術から、オルガノイドおよび心筋細胞に分化した誘導多能性幹細胞の使用に至るまで、大きな進歩を遂げた1,2,3。エクスビボおよび初代細胞培養は、特に遺伝学的研究および創薬のために、大きな価値があることが示されている4,5,6。ヒト組織を使用すると、結果の翻訳価値が向上します。しかし、無傷の形状を有する心筋組織の長期3D培養は十分に確立されていない。インタクトな幾何学的形状は、適切な心機能、異なる細胞間のコミュニケーション、ならびに細胞 - マトリックス相互作用が前提条件であるため、in vivo状態を模倣するための重要な特徴である。心筋組織培養は、発達の様々な段階を経た。エクスビボ心筋組織培養の成功率および安定性は、当初はかなり低かったが、最近のアプローチは有望な結果7、8、9、10、11をもたらしている。
その中で、Fischerらは、ヒト心筋組織の生存率および収縮性能が、エキソビボ細胞培養において何週間も維持され得ることを最初に実証した7。彼らの技術は、外植されたヒト心筋から切断された薄い組織スライスに基づいており、それらは、定義された生体力学的条件および連続的な電気刺激を提供する新しく開発された培養チャンバーに取り付けられた。この培養方法は、心筋組織のin vivo機能によく似ており、いくつかの独立した研究グループ2、12、13、14、15によって再現されている。重要なことに、Fischerらが使用したチャンバーは、最大4ヶ月間、発達した力の継続的な登録を可能にし、したがって、無傷のヒト心筋に関する生理学的および薬理学的研究のための前例のない機会を開いた7。
同様の技術を他のグループによって独立して開発し、ヒト、ラット、ブタ、およびウサギ心筋に適用した7、10、11。Pitoulisらはその後、収縮サイクル中の法線力-長さ関係を再現するより生理学的方法を開発したが、ハイスループット解析には適していない16。このように、生物模倣体培養の一般的なアプローチは、動物実験の還元、改良、および置換(3R)へのさらなるステップと見なすことができる。
ただし、この可能性を活用するには、標準化された手順、高コンテンツ分析、および高スループットレベルが必要です。我々は、市販されている生体模倣体培養システムにおいて、生きたヒト心筋の自動スライスと in vitro 維持を組み合わせた技術を提示する( 材料表参照)。提案されたアプローチでは、単一の経壁心筋標本から生成することができる個々のスライスの数は、処理時間によってのみ制限される。十分なサイズと品質(3 cm x 3 cm)の標本は、多くの場合、自動ビブラトームで便利に切断される20〜40の組織スライスを生成します。これらのスライスは、システムに属する培養チャンバに入れることができる。チャンバは、チャンバ内のスプリングワイヤを使用して、変調可能なパラメータ(すなわち、パルス持続時間、極性、レート、および電流)ならびに前負荷および後負荷の調整が可能な電気刺激を可能にする。各スライスの収縮は、ばね線に取り付けられた小さな磁石の動きから登録され、解釈可能なグラフとして表示されます。データはいつでも記録でき、自由に利用できるソフトウェアを使用して分析することができます。一定のベースラインペーシングとは別に、スケジュールされたプロトコルを実行して、それらの耐火性期間、刺激閾値、一時停止後増強、および力 - 周波数の関係を機能的に評価することができる。
個々の心臓から複数の心筋スライスを培養するこの長期の生体模倣体は、ヒトおよび動物組織の両方における将来のエクスビボ研究への道を開き、心血管内科における治療的および心毒性薬物効果のスクリーニングを容易にする。既に様々な実験的アプローチ2、12、13、15に適用されている。ここでは、ヒト組織の調製について詳細に段階的に説明し、頻繁に遭遇する培養上の問題に対する解決策を提供する。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Low melting point</td>
			<td>Roth</td>
			<td>6351.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bay-K8644</td>
			<td>Cayman Chemical</td>
			<td>19988</td>
			<td></td>
		</tr>
		<tr>
			<td>BDM (2,3-Butanedione monoxime)</td>
			<td>Sigma</td>
			<td>B0753-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2*H2O</td>
			<td>Merck</td>
			<td>2382.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Calciseptine</td>
			<td>Alomone Labs</td>
			<td>SPC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose*H2O</td>
			<td>AppliChem</td>
			<td>A3730.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>BBraun</td>
			<td>3703452</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem</td>
			<td>A1069.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoacryl</td>
			<td>BBraun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol 100%</td>
			<td>SAV LP GmbH</td>
			<td>UN1219</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS-X-supplement</td>
			<td>Gibco</td>
			<td>5150056</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04933.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Gibco</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2*6H2O</td>
			<td>AppliChem</td>
			<td>A1036.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4*H2O</td>
			<td>Merck</td>
			<td>1.06346.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine</td>
			<td>Sigma</td>
			<td>N7634-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin x100</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>AppliChem</td>
			<td>A1108.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cabinet</td>
			<td>Thermo Scientific</td>
			<td>KS15</td>
			<td></td>
		</tr>
		<tr>
			<td>Frigomix waterpump and cooling + BBraun Thermomix BM</td>
			<td>BBraun</td>
			<td></td>
			<td>In-house made combination of cooling and heating solution.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>CB240</td>
			<td></td>
		</tr>
		<tr>
			<td>MyoDish bioreactor system</td>
			<td>InVitroSys GmbH</td>
			<td>MyoDish 1</td>
			<td>Myodish cultute system</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200s</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath 37 degrees</td>
			<td>Haake</td>
			<td>SWB25</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath 80 degrees</td>
			<td>Daglef Patz KG</td>
			<td>7070</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL plastic single-use beaker</td>
			<td>Sarstedt</td>
			<td>75.562.105</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration unit, Steritop Quick Release</td>
			<td>Millipore</td>
			<td>S2GPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 0.9 x 70 mm 20G</td>
			<td>BBraun</td>
			<td>4665791</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic triangles</td>
			<td></td>
			<td></td>
			<td>In-house made</td>
		</tr>
		<tr>
			<td>Razor Derby premium</td>
			<td>Derby Tokai</td>
			<td>B072HJCFK6</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Gillette Silver Blue</td>
			<td>Gillette</td>
			<td>7393560010170</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel disposable</td>
			<td>Feather</td>
			<td>02.001.30.020</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL Luer tip BD Discardit</td>
			<td>BBraun</td>
			<td>309110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 10 cm</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 3.5 cm</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of human myocardial tissue for long-term cultivation

心筋細胞培養は、2次元(2D)細胞培養からiPSC由来オルガノイドまで、膨大な数の開発を見てきました。2019年には、心筋収縮のインビボ状態に近づきながら、ヒト心臓サンプルから得られた心筋スライスを培養するエクスビボ方法が実証されました。これらのサンプルは、主に心臓移植または左心室補助装置の配置に由来する。ビブラトームと特別に開発された栽培システムを使用して、固定ワイヤとスプリングワイヤの間に厚さ300μmのスライスを配置し、数週間安定して再現可能な栽培を可能にします。栽培中、スライスは個々の設定に従って連続的に刺激される。収縮はリアルタイムで表示および記録することができ、薬理学的薬剤を容易に適用することができる。ユーザー定義の刺激プロトコルをスケジュールして実行し、一時停止後増強、刺激閾値、力と周波数の関係、耐火期間などの重要な収縮パラメータを評価することができます。さらに、このシステムは、より生理学的な培養のための可変のプリロードおよびアフターロード設定を可能にする。
ここでは、市販の生体模倣培養溶液を用いて、ヒト左心室心筋スライスの長期培養を成功させる方法に関するステップバイステップガイドを提示する。

心筋スライスの収縮は、培養チャンバを対応するコネクタに挿入した後、コンピュータ画面に表示された(図3)。ヒト心筋切片の収縮は、刺激直後に開始された。スライスは5〜10分間過収縮した。これは、損傷組織画分の強直拘縮によって引き起こされる拡張期力の増加として見られた。このプロセスは、1〜1.5時間以内に様々な程度に戻された。安定化後、ヒトLV組織ス?...

Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

Preparation of Human Myocardial Tissue for Long-Term Cultivation

我々は、ヒト心室心筋組織の エクスビボ 培養のためのプロトコールを提示する。これは、収縮力および動態の詳細な分析、ならびに インビボ 生理学的環境をより密接に模倣するための前負荷および後負荷の適用を可能にする。

長期培養のためのヒト心筋組織の作製

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

このプロトコールを用いて、ヒト左心室組織切片を生体模倣チャンバー内でエキソビボで培養することができる。前負荷と後負荷の適用は、生理学的環境の類似性を改善する。この技術は、組織収縮の連続的な登録を可能にする。ユーザー定義の刺激プロトコルにより、一時停止後の増強、刺激閾値、力と周波数の関係、耐火期間などの重要な収縮パラメータの評価が可能になります。このセットアップを使用した心筋スライスの長期培養は、将来のエクスビボ研究への道を開き、心血管医学における治療的および心毒性薬物効果のスクリーニングを容易にする。まず、培養チャンバとグラファイト電極を1リットルの10%イソプロパノール溶液に沈め、一晩攪拌します。翌日、チャンバを100%イソプロパノール溶液に3分間移し、チャンバおよびグラファイト電極を層流フードの下で風乾させる。ロッカーの使用可能な位置に応じて、各チャンバーに回路基板を取り付けます。製造元の指示に従って回路基板に2つのグラファイト電極を置き、感染を防ぐために35ミリメートルのペトリ皿の蓋をチャンバーに置きます。次に、切断トレイをスライスバッファーで最大90〜95%まで満たします。組織サンプルをコールドスライスバッファーで満たされた100ミリメートルのペトリ皿に移し、皿を摂氏4度の冷却プレートの上に置きます。ピンセットで心内膜を保持して心内膜線維柱帯を除去し、はさみを使用して心内膜組織の約3ミリメートルを切り取る。切断した組織サンプルを、正方形の位置に固定された4本の0.9 x 70ミリメートルの20ゲージ針を使用して、心内膜側を2 x 2 cmのゴムパッチまで固定します。各針先の対角線の端が内側を向いていることを確認し、固定を強化し、心筋の損傷を防ぎます。メスを使用して、4つの針の正方形の外側にある余分な組織をすべて切り取ります。ピンセットを使用して、トリミングしたサンプルを滅菌組織片に10秒間置き、余分なスライスバッファーを除去します。次に、アガロースシリンジをウォーターバスから取り出し、サンプルをアガロースに沈める。アガロースを冷却プレート上で5分間固化させます。サンプルの心内膜側はアガロースで見えなければなりません。ピンセットを使用して、アガロースに含まれるサンプルの心外膜側を接着領域の上に置き、アガロースを切断または損傷することなく、鈍い工具でアガロース含有サンプルを上から静かに押します。接着剤を1分間固めます。振動振幅を1ミリメートル、初期切削速度を毎秒0.07ミリメートル、スライスの厚さを300ミクロンに設定します。栽培システムをコンピュータに接続した後、対応するソフトウェアプログラムを起動します。ロッカーの速度を60rpmに設定し、刺激パラメータをプリセットします。ヒトの心臓スライスの場合、標準刺激を、50ミリアンペアまたは3ミリ秒の正電流、1ミリ秒の一時停止、および3ミリ秒の反転電流パルスからなる電流の二相性インパルスに設定し、毎分30ビートまたはbpmのペーシングレートで、ソフトウェアの電極インジケータをチェックし、栽培室の電極が正しく機能していることを確認します。ピンセットを使用して、アガロースを組織から分離する。組織に触れないようにし、組織への損傷は栽培の成功率を低下させるので慎重に取り扱います。サンプルに2つのプラスチック製の三角形を取り付けるには、滅菌ペトリ皿の蓋に1マイクロリットルの接着剤を置きます。フックピンセットを使用して、オートクレーブ処理されたプラスチック製の三角形を1つ拾います。三角形の前端を接着剤に素早く浸し、心筋細胞のアライメントに垂直なサンプルに三角形を貼り付けます。もう一方の三角形についても同じ手順を繰り返します。メスを使用して、三角形の幅を超える組織をトリミングし、2つの三角形が取り付けられたスライスをスライスバッファーを含む切断トレイに戻します。培地充填培養チャンバーをインキュベーターから取り外します。用意したスライスを1つ選択し、各ピンに1つの三角形を接続してチャンバに挿入します。取り付けピン間の距離をサンプルサイズに合わせて調整します。サンプルが培地に沈んでいることを確認します。皿をロッカーに置いた後、コンピュータ画面上の対応するグラフのベースラインがもう変わらなくなるまで調整ネジを反時計回りに回して予荷重を下げ、調整ネジを時計回りに回して予荷重または張力を慎重に上げます。剛性の高いチャンバの場合、グラフ内の対応するベースラインが 1 ミリニュートンの予荷重に対応する 1,000 ~ 1,200 単位増加するまで続けます。新鮮な栽培培地を調製した後、37°Cのウォーターバスまたは熱風インキュベーターで培地を30〜45分間予備温める。チャンバーから培地を取り出し、チャンバー内に約0.8ミリリットルを残してから、同じチャンバーに1.6ミリリットルの新鮮な培地を加え、チャンバーあたり2.4ミリリットルの培地の総容量を作ります。最後に、チャンバーのカバーを背面に置き、栽培チャンバーをそれぞれの位置に置きます。典型的な刺激中の5つの心筋スライスの収縮の読み出しは、一時停止後の増強、刺激閾値、力 - 周波数関係、および耐火期間の4つの異なるセクションから成っていた。対照と比較して、カルシウムアンタゴニスト、ニフェジピンおよびカルシセプチンで処理したスライスの収縮力は、10分以内に減少した。対照的に、電位依存性カルシウムチャネルアゴニストBay-K8644は、収縮力を増加させた。対照および処置切片の休止後増強を、筋小胞体からの細胞内カルシウム放出について評価した。コントロールスライスに変更が表示されませんでした。カルシセプチンおよびニフェジピンの存在下では、L型カルシウムチャネルの阻害は、50秒の一時停止後の最初の収縮の増強をもたらし、全収縮性に対する細胞内カルシウム放出のより高い相対的寄与を反映した。反対の効果は、L型カルシウムチャネルを介した細胞外カルシウムの侵入を刺激するBay-K8644で観察された。力-頻度関係解析では、コントロールスライスに変化は認められなかった。カルシセプチン処置は、処置前および治療後のデータを比較したときの刺激頻度の増加時に刺激に従うスライスの能力を変化させなかった。カルシセプチンと比較して, ニフェジピンは、より高いペーシング速度で収縮性の増加を防止し、で最大捕捉率を低下させました 80 bpm.Bay-K8644では、非常に低い刺激周波数で収縮力の増加が観察された。しかし、50bpmより高い周波数では、収縮力は前処理条件時よりも低かった。摘出した組織は、速やかに4度の心筋麻痺に移すべきである。スライス後、プラスチック三角形をファイバー方向に垂直に取り付ける必要があります。プリロードは1500ミリニュートンを超えてはなりません。生体模倣スライス培養は、研究者が成人ヒト心筋の再生と修復を研究するための遺伝子操作および細胞ベースの治療法を利用するのに役立ちます。

preparation-of-human-myocardial-tissue-for-long-term-cultivation

Research

JoVE Journal

Medicine

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

内皮角膜移植のために角膜ドナー組織の準備

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

医学

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

トランスレーショナルがん研究のためのヒト組織での作業

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

新生豚のリンパ節の腸リンパトランクやコレクションの長期カテーテル法

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

豚の長期心血管運動テストのためのカテーテルの外科的配置

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

テレメトリを使用して自由に動くマウスにおける長期血圧測定

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3Dホール・ハート心筋組織の分析

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

組織と血液サンプリング手順の標準化による閉胸ブタ心筋梗塞モデルの改善

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

マウスの不整脈を検出するための長期心電図記録の分析

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

脂肪組織から間葉系幹細胞を得る技術および長期凍結保存における間質血管画分のキャラクタリゼーション

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).