This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

このプロトコルは、動物実験倫理委員会のLUMCのガイドラインに従っています。 楽器の調製、培養培地、及びMTCS 注：層流フード内のすべての準備を行います。 MTCS灌流チャンバー、気泡トラップとスタンドリーバーら 、2010 11に記載されています。 <ol><li> 70％エタノールで鉗子とマイクロはさみを消毒します。無菌タイロード緩衝液（130mMの塩化ナトリウム、5.4のKCl、6.0 mMのヘペス、1.2 mMの硫酸マグネシウム、1.2 mMのKH 2 PO 4、20mMのグルコース、1.5mMの塩化カルシウム2・2H 2 Oで21G針で5 mlシリンジを準備pH値= 7.2）。無菌KCl溶液（100 mM）ので21G針で5 mlシリンジを準備します。 </li><li>心あたりのメディアの65ミリリットルを準備し、10％ウシ胎児血清を補充したDMEM、抗生物質/抗真菌剤（A / A、ペニシリン100単位、ストレプトマイシン100μgの、およびアムホテリシンB / mlを0.25 UG）、インスリントランスフェリンセレン（ITS;を10μg/ mlのインスリン、5.5 / mlのトランスフェリン、6.7 ngの/ mlの亜セレン酸ナトリウム）、フィルター滅菌します。 </li><li> 70％エタノール、紙タオルで乾燥して噴霧することにより、灌流チャンバー及び気泡トラップを滅菌します。最初に灌流チャンバーなしに、しかし、MTCS（ 図1D）を組み立てます。スタンドに気泡トラップを配置します。簡単に負荷のポンプヘッドと蠕動ローラーポンプを使用してください。 <ol><li>シリコンチューブを使用すると、リザーバ（50mlチューブ）、ポンプ、ポンプおよび気泡トラップ、気泡トラップとリザーバ（ 図1Dを参照）との間の接続を行います。媒体が気体透過性のシリコンチューブ（1メートル）とできるだけ短くインキュベーター外管を通してインキュベーターでガスでガス平衡に到達することを可能にする十分な長さインキュベーター内部の配管をしてください。 </li></ol></li><li> 、70％エタノールで50mlのチューブを埋める（適切な循環を可能にするために、1ml /分の周りの流速）は、ポンプをオンにし、エタノールのCIRを聞かせてすべてのチューブを滅菌するために30分間culate。貯蔵器を空にし、システムからエタノールをポンピングすることにより、エタノールを除去します。 </li><li> 、滅菌蒸留水で50mlのチューブを埋めるポンプをオンにして、水が残っているエタノールを取り除くために30分間循環させて。貯蔵器を空にし、システムから水をポンプで水を除去します。 </li><li>メディアの45ミリリットルと50ミリリットルチューブを埋める、インキュベーター中にスタンドを置きます。ポンプ（1 ml /分の周りの流速）をオンにして、すべてのチューブを埋めるための媒体を循環させ、すべての気泡を除去し、培地は、少なくとも1時間インキュベーター内ガス組成に適応することができます。標準的な組織培養条件（5％CO 2、37℃）でインキュベーターを維持します。気泡がチューブ内に存在していないことを確認してください。 </li></ol>マウスの心臓の2の単離<ol><li>ヘパリン500単位でマウスを注入します。これは、凝固血液を防止し、Bの除去を可能にするであろう心の底からlood。 10分後、精密気化器を用いて、4％イソフルランで誘導チャンバ内でマウスを麻酔。マウスはつま先のピンチに応答しない時の麻酔で十分です。 </li><li>解剖ボードにマウスを移し、同軸回路に接続されたフェイスマスクを用いて麻酔を維持します。 70％のアルコールでマウスの毛を消毒します。はさみは、腹腔を開いて使用すると、大静脈及びダイヤフラムを露出させます。 </li><li>心臓を露出するために、最後から最初の肋骨に起動横切開を行い、マウスの頭の上に胸郭を反映しています。マウスはもう呼吸していないされているように麻酔を停止します。心臓の鼓動を観察します。 </li><li> （ダイアフラムを介して）胸腔内に腹部から下大静脈に無菌タイロード緩衝液を含む5 mlシリンジに取り付けた21G針を挿入します。血液が針の挿入から大静脈の尾側から出ることができることを確認してください。目を灌流電子タイロード緩衝液を静かにと大静脈への一定の圧力で心臓の一部が、その赤い色を失うまで。注意：心臓は、心臓からの血液の除去を可能鼓動のまま。 </li><li>無菌のKCl溶液を含む5 mlシリンジに取り付けた21G針で針を交換してください。静かに心臓が鼓動を停止するまで大静脈にKCl溶液を灌流し、針を取り除きます。注意：KCl溶液を灌流針と冠動脈系の灌流を容易に挿入できるようになりますリラックスした拡張期に心を保持します。 </li><li>わずかに湾曲した鉗子を用いて心臓を持ち上げ、ハサミを使用して、周辺組織から自由に心を分析無傷と心臓に接続されている心臓の近位に動脈と静脈を残します。氷冷PBSを含む15mlチューブに心を移し抗生物質/抗真菌剤（A / A）を補いました。最大3時間、氷冷PBS中で心を保管してください。 </li></ol> 3. Cannulatio灌流チャンバー内にマウスハーツのN <ol><li>層流フード内のすべての手順を実行します。 10センチメートルシャーレに15mlチューブから分離された心を移し、PBSを追加し、A / Aを補いました。 </li><li>解剖顕微鏡下では、すべての非心臓組織を除去するために、マイクロはさみとピンセットを使用していますが、上行大動脈を維持する心臓に腕頭動脈と肺静脈、2mmの近位との少なくとも分岐します。左心室の内腔（通常は2ミリメートル）へのアクセスを作成するために、心尖の先端をカット。解剖顕微鏡下流フードで灌流チャンバーを配置。 </li><li>シリコンチューブを使用して、挿入針に培地で5 mlシリンジを取り付けます。培地20mlで灌流チャンバーを記入し、灌流チャンバー（ 図1）で2平滑化した針との間で回転ステージに心を置きます。心臓は、目の前に配置されるように、回転ステージの高さを調整します針。 </li></ol>僧帽弁を培養するための4ライゲーション（図1参照） <ol><li>縫合糸（絹7.0）で大動脈を連結。縫合糸で左心耳を結紮。左心房への肺静脈を通って（図1に1をNR）針を挿入し、左心房内のエントリに縫合糸の近位で連結します。これは、僧帽弁に損傷を与えるよう針が左心房にすぎないことを確認します。 </li><li>左心室に直線運動で針（ 図1の nr.2）を挿入します。 50mlのチューブに灌流チャンバーからメディアを転送します。生体適合性の接着剤で心筋に針をシール。 </li><li>接着剤が乾燥した後、慎重にnr.1針や漏れがあるかどうかを確認するための媒体で満たされた注射器を接続することにより、心に培地を注入します。針nr.2と最後に残った血液からの媒体出口は心の外に灌流されていることを確認します。 </li></ol> 5.リー大動脈弁を培養するためのゲーション（図1を参照） <ol><li>大動脈に注射針（ 図1の nr.1）を挿入し、縫合糸で連結します。これは大動脈弁を損傷するよう針が大動脈にすぎないことを確認します。左心室に直線運動で針（ 図1の nr.2）を挿入します。 50mlのチューブに灌流チャンバーからメディアを転送します。生体適合性の接着剤で心筋に針をシール。 </li><li>接着剤が乾燥した後、慎重にnr.2針や漏れがあるかどうかを確認するための媒体で満たされた注射器を接続することにより、心に培地を注入します。針nr.1と最後に残った血液からの媒体出口は心の外に灌流されていることを確認します。 </li></ol>スタンドに6.灌流チャンバー<ol><li>メディア転送20mlで灌流チャンバーを埋めます。室にガスケットと蓋を置き、ワッシャー、ネジで締めます。 </li><li> incubaから組み立てMTCSを削除TOR。スタンドに灌流チャンバーを取り付けます。チューブを接続します（図1Dを参照してください ）。インキュベーター（5％CO 2、37℃）にスタンドを置きます。ポンプにチューブを接続します。 600μL/分の周りの流速でポンプの電源を入れます。 </li><li>貯留および/または気泡トラップ媒体のレベルが入ってくるメディア滴の落下によって流れの存在の視覚化を可能にすることを確認します。培養後、心臓は、システムから除去固定し、組織学的検査のために処理されます。 </li></ol>

<ol>
	<li>Go, A. 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=Heart+Disease+and+Stroke+Statistics--2014+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2014 Update: A Report From the American Heart Association.</a> Circulation. 129, e28-e292 (2013).</li><li>Gould, S. T., Srigunapalan, S., Simmons, C. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+and+Cellular+Response+Feedback+in+Calcific+Aortic+Valve+Disease.">Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease.</a> Circulation Research. 113 (2), 186-197 (2013).</li><li>Kruithof, B. P. T., Krawitz, S. A., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrioventricular+valve+development+during+late+embryonic+and+postnatal+stages+involves+condensation+and+extracellular+matrix+remodeling.">Atrioventricular valve development during late embryonic and postnatal stages involves condensation and extracellular matrix remodeling.</a> Developmental biology. 302 (1), 208-217 (2007).</li><li>Schoen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+valves+and+valvular+pathology:+update+on+function,+disease,+repair,+and+replacement.">Cardiac valves and valvular pathology: update on function, disease, repair, and replacement.</a> Cardiovascular pathology: the official journal of the Society for Cardiovascular Pathology. 14 (4), 189-194 (2005).</li><li>Balachandran, K., Sucosky, P., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+mechanobiology+of+aortic+valve+inflammation+and+calcification.">Hemodynamics and mechanobiology of aortic valve inflammation and calcification.</a> International journal of inflammation. , 263870 (2011).</li><li>Butcher, J. T., Simmons, C. A., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+the+Aortic+Heart+Valve.">Mechanobiology of the Aortic Heart Valve.</a> The Journal of heart valve disease. 17 (1), 62-73 (2008).</li><li>Balachandran, K., Konduri, S., Sucosky, P., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+study+of+the+biological+properties+of+porcine+aortic+valves+in+response+to+circumferential+cyclic+stretch.">An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch.</a> Annals of biomedical engineering. 34 (11), 1655-1665 (2006).</li><li>Weiler, M., Hwai Yap, C., Balachandran, K., Padala, M., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+analysis+of+dynamic+deformation+characteristics+of+native+aortic+valve+leaflets.">Regional analysis of dynamic deformation characteristics of native aortic valve leaflets.</a> Journal of Biomechanics. 44, 1459-1465 (2011).</li><li>Combs, M. D., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+Regulatory+networks+in+development+and+disease.">Heart valve development: Regulatory networks in development and disease.</a> Circulation Research. 105, 408-421 (2009).</li><li>Kruithof, B. P. T., Duim, S. N., Moerkamp, A. T., Goumans, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;+and+BMP+signaling+in+cardiac+cushion+formation:+lessons+from+mice+and+chicken.">TGF&#946; and BMP signaling in cardiac cushion formation: lessons from mice and chicken.</a> Differentiation; research in biological diversity. 84 (1), 89-102 (2012).</li><li>Lieber, S. C., Kruithof, B. P. T., Aubry, N., Vatner, S. F., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+miniature+tissue+culture+system+to+culture+mouse+heart+valves.">Design of a miniature tissue culture system to culture mouse heart valves.</a> Annals of biomedical engineering. 38 (3), 674-682 (2010).</li></ol>

The authors declare no conflicts of interest.

マウス心臓弁の培養における重要なステップは、流れの適切な向きを確保するために、マウスから心臓の切除および弁に対して垂直に針の生存率および連結を確実にするためにできるだけ短く灌流チャンバー内ライゲーションの間の時間を作る挙げ。さらに、メディアなし灌流チャンバー内ライゲーション後の流れをチェックする適切な挿入や針の連結を確実にします。これは、無菌培養を?...

心臓弁疾患は、西洋世界における罹患率および死亡率の主要な原因です。その有病率は年齢とともに増加し、それは人口75年の10％以上と1古い影響を与えます。ハートの全身一部、大動脈弁と僧帽弁の弁は、ほとんど影響を受けています。心臓弁疾患は、機械的特性2の変化をもたらすバルブの高度に組織構造の喪失によって特徴づけられます。構造的完全性は、弁の機能のために極めて重要です。 弁のリーフレットは、非常に層状のパターン3,4で構成されて弁膜間質細胞（VIC）、弁膜内皮細胞（VEC）、および細胞外マトリックス、から構成されています。 VICには、ECM合成、分解と組織を担当しています。要因としては、血流から発せられる電気部品やその機能を組織のVICにECMの行為に常駐しています。加えて、機械的な力は、圧縮または引張応力がVICの5の動作に影響を与える、層状または振動せん断応力が生じる心周期中のリーフレットに作用します。 弁の構造が制御されている方法を理解するためには、最初のVICは、心周期の間に経験した刺激の多様なセットにどのように反応するかを理解する必要があります。in vitro試験は、心臓弁膜細胞の特性や能力について非常に有益でした。 in vitroでのこれらの細胞の応答は、しかし、常に正確にin vivoでの応答6を模倣しないことがあります。例えば、刺激にVICの応答は、ECおよびECM組成5の存在に依存します。また、刺激に対する弁膜細胞の応答は、リーフレット7に、その特定の場所に依存します。生化学的な刺激に加えて、弁の細胞の挙動は、Oに作用する機械的な力によって決定されますn個のバルブ8。バルブの各領域は、血行力学的応力の自身の特定のセットに供されます。現在のex vivoでのモデルは、機械的な力が弁膜構造5の重要な決定因子であることを示してきましたが、関連するメカニズムはまだ不明です。 in vivoモデルは、弁膜開発9,10の根底にある分子メカニズムへの洞察を提供してきたが、大人弁膜生物学への洞察はまだとらえどころのないです。 したがって、ex vivoでのフロー・モデルとは、心臓弁は、時間11の長期間の心に彼らの自然な位置で培養することができる開発されました。これは、弁がその天然のコンフィギュレーションに残りおよびVICは、VICの応答が可能な限り自然な刺激になって、 インビボのと同じ環境を経験するという利点を有します。また、心の中で彼らの自然な位置にある弁の文化はそれぞれを施す容易関連する血行動態ストレスに弁膜地域。このex vivoでのモデル、 すなわち、小型組織培養系（MTCS）において、弁は、心臓弁のリモデリングにおけるそれらの役割の研究を可能にする種々の生化学的及び血行力学的な刺激を受けることができます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>life technolgies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>life technolgies</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium</td>
			<td>life technolgies</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>life technolgies</td>
			<td>15240-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk 7-0</td>
			<td>Ethicon</td>
			<td>768G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dish</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe</td>
			<td>BD</td>
			<td>309649</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>BD</td>
			<td>304432</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>LEO</td>
			<td>012866-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>Tedpella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>Thermo Scientific</td>
			<td>8060-0020</td>
			<td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td>
		</tr>
		<tr>
			<td>Silicon tubing for pump</td>
			<td>Masterflex</td>
			<td>96400-13</td>
			<td>I.D. x O.D. x Wall: 0,8 x1,59 x 0,40 mm</td>
		</tr>
		<tr>
			<td>Biocompatible glue (Histoacryl)</td>
			<td>B. Braun</td>
			<td>1050071</td>
			<td></td>
		</tr>
		<tr>
			<td>precision vaporizer</td>
			<td>Dr&#228;ger</td>
			<td>Vapor 200</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic roller pump</td>
			<td>Masterflex</td>
			<td>7521-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-load pump head</td>
			<td>Masterflex</td>
			<td>7518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow chamber</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
	</tbody>
</table>

culturing mouse cardiac valves in the miniature tissue culture system

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 molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

大動脈弁（ 図2）または僧帽弁11には、少なくとも 3日間培養することができます。 （大動脈弁と僧帽弁用拡張期位置のための収縮期位置を表す）開位置に培養することにより、弁膜細胞が生存したまま。 TUNEL陽性細胞（ 図2H、I）、または切断されたカスパーゼ3の発現の欠如によって決定されるように何らの細胞死が観察されない（図示していないリーバー<...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

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 ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

Research

JoVE Journal

Bioengineering

8.8K ビュー.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

ビデオ: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

タンパク質分解的に分解性3次元システムにおける卵胞のカプセル化と文化のための方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

生物工学

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

心臓組織工学のためのフィブリンゲルにおける心筋細胞のカプセル化

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

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 ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

灌流固定標本のコントラストコンピュータ断層撮影を用いた人間の心臓静脈系の解剖学的再建

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

トライリーフレットポリマーバルブの相対的な流体力学的評価のためのプロトコル

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

人間の3次元の組織工学<em&gt; in vitroで</em&gt;腫瘍テストシステム

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

アン<em&gt;インビトロ</emマウス椎間板の&gt;器官培養モデル

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

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 ...

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

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

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

スキャフォールド フリー三次元心筋組織構築純金型ベース方式

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