著者らは、研究の最初の部分のための財政支援のための教授ヘンドリックTevaearaiと心臓血管外科に感謝します。

注：ルイス雌雄ラット、200〜220グラムは、標準的な実験室条件（12時間の明暗周期、 自由摂取させ 、 水や食料、IVCケージ）の下で飼育した。すべての動物は、動物保護に関するFELASAとスイス法の勧告に準拠して処理した。 1。細胞の調製：骨髄から間葉系幹細胞の単離<ol><li> 5分間の誘導チャンバ内でイソフルラン5％とO 2を5L /分でラットを麻酔。麻酔システムに接続されたノーズコーンに動物の鼻を置きます。鎮静を確認するために、足指や尾のピンチを実行します。 </li><li>腰に足首から皮膚をカットしてはさみで足から皮膚を取り除きます。筋肉を外し、動物失血のための大腿動脈を切った。股関節を公開し、大腿骨頭を脱臼。これは、大腿骨頭に損傷を与えないことが重要である。 50ミリリットルのプラスチックチューブに滅菌PBS中の骨を置きます。 </li><li>すべてのムーを削除それらを壊すことなく骨からsclesや靭帯。注：骨エタノールで洗浄溶液または汚染の連鎖を避けるために無傷のままであることが重要である。 5分間、70％エタノール中で剥離した骨を洗い流す。 </li><li>無菌層流フードの下では、はさみで骨の両方の先端をカット。骨端から滅菌PBSを注入することにより、骨髄をフラッシュします。 15ミリリットルチューブに骨髄を収集します。 300×gで遠心し7分。 </li><li>上清を取り除きます。赤血球溶解バッファー3mlにペレットを一時停止します。 300×gで7分間再びスピンする前に室温で1分間、懸濁液をインキュベートする。 150ミリリットルの培養フラスコ中で、無菌細胞培養培地（395ミリリットルIMDM培地を5mlのペニシリン/ストレプトマイシンを100mlのFBS）を添加し、細胞を播種する。 </li><li>非付着細胞を除去するために二日目の媒体に変更します。 <ol><li>所望の細胞量が得られるまで2〜3週間おきに毎日培地交換を行います。 </li><li>代わりに、種子によってbiograftを準備必要に応じて他の場所3記載の固体マトリックス上に細胞をる。 </li></ol></li><li>心臓移​​植の日には、細胞を調製または単に心外膜適用前にbiograftを収穫。 <ol><li>のAccutase細胞剥離液を用いて細胞を収集します。細胞を数える。所望の細胞量を得るために計算された細胞懸濁液の体積を1.5 mlの遠心チューブを埋める。 300×gで7分間遠心分離し、上澄みを除去します。 </li><li>あるいは、滅菌PBSですすぎ、細胞培養培地からbiograftを採取し、血清を含まない新鮮な培地中で維持する。 </li></ol></li></ol>まず開胸とLAD結紮<ol><li>ラットを計量。 37℃で加熱パッドをオンにします。 5-7分間の誘導室中の5％イソフルランおよび5 L /分、100％O 2でラットを麻酔。鎮静を確認するために、足指や尾のピンチを実行します。加熱パッド上にラットを置きます。 </li><li> 14を動物に挿管G静脈カテーテル。 2.5リットル/分の酸素、2.5％イソフルラン、2ミリリットルの一回換気量、および90呼吸/分の呼吸頻度のためにプログラムさ齧歯類ベンチレータに挿管カテーテルを接続します。 </li><li> 0.1ミリグラム/ kgのブプレノルフィン溶液を調製する。液の皮下に三分の一を注入。胸部の左側の部分を剃る。 1％ベタジン溶液で消毒。 </li><li>第四肋間で胸骨に垂直に皮膚を切開。胸の筋肉（大胸、胸を昇順、および外腹斜胸筋）の3層を分離。 （リブ4と5の間）第四肋間を開きます。 </li><li>肋骨を広げるためにと心を露出させ、小さなリトラクターを使用してください。慎重に心膜を開きます。アトリウム以下の7.0縫合糸4ミリメートルで冠状動脈（LAD）を左前の位置を確認し、連結する。 </li><li> 3.0縫合を使用して2針で肋間スペースを閉じます。胸骨から近位および遠位に2本の縫合糸を配置します。まず、T遠位縫合糸をighten。胸を膨らませると、任意の気胸を避けるために、2秒間の人工呼吸器の排気チューブをクランプします。第2の縫合糸を締めます。 （無縫合は必要ありません）が所定の場所に戻り、筋肉層を配置します。 5.0縫合で皮膚を閉じます。 </li><li> 1％ベタジン溶液で縫合糸を駆除。ブプレノルフィン液の残りを注入します。麻酔システムの電源をオフにします。挿管カテーテルを取り外します。 </li><li> 1〜2時間、暖かいランプの下にケージにラットを保管してください。ケージの中に温度計を保持し、過熱を避けるために、温かいランプとケージとの間の距離を制御します。 IVCユニットにラットを返します。 48時間皮下ブプレノルフィン0.05〜0.1ミリグラム/ kgのポスト-operativelyごとに6〜12時間後に注入する。 </li></ol>第二に開胸処理によって3。心外膜の管理<ol><li>繰り返します2.1から2.3を繰り返します。 </li><li>第五肋間で胸骨に垂直に皮膚を切開。 3層を分離胸の筋肉（大胸、昇順胸筋と外腹斜胸筋）のsの。 （リブ5と6の間）第五肋間を開きます。 </li><li>肋骨を広げるためにと心を露出させ、小さなリトラクターを使用してください。いくつかの付着がある場合は、慎重に細かい鉗子でそれらを削除します。 </li><li>合字下の淡い領域として表示される梗塞領域の位置を確認します。注：必要に応じて、より良い左心室を可視化するために頂点に挿入された縫合糸の10cmのピース（7.0）を使用します。心臓を露出させクランプによって緩く維持縫合糸に慎重に引き抜いてください。 </li><li>以下の処置のいずれかを適用します。 <ol><li>心臓の表面にbiograftを配置します。 10分間室温で予め温めプレフィルドフィブリンシーラント注射器を使用してください。 25 Gルアーの針を用いてbiograft下のフィブリンシーラントの低下（50〜100μl）を適用します。 biograftが完全に密封されていることを確認します。 </li><li>あるいは、表面oをでピペットの先端で収集した細胞ペレットを適用する合字下の淡い領域に心F。細胞ペレット上にフィブリンシーラントの低下（50〜100μl）を適用します。 </li></ol></li><li>頂点から縫合糸の部分を削除します。 </li><li>繰り返します2.6から2.8を繰り返します。 </li></ol>

<ol>
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The authors have nothing to disclose.

LADの永久的な連結は、不可逆的な心筋障害を引き起こす。最初の動物モデルは、1960 4に記載した。以来、それは、慢性MIのための標準および適切なモデルとして考えられてきた。その安定性と再現性は、MI 5のための治療法の実験的評価を可能にした。最初の説明の後の改善の手順は、35から13までパーセント6の手術死亡率を報告した。 予想され?...

19 世紀末以来、心血管疾患は、先進国でナンバーワンの連続殺人犯を推移している。これらの中でも、冠動脈疾患、メイン病因を表す。心筋梗塞（MI）および急性期の結果は次第に慢性期および重症心不全に向かって発達不適応リモデリングが続く。最近の重要な技術的および治療 ​​の進歩にもかかわらず、原因心不全の進行に罹患率と死亡率は依然として1を拡大しています。この文脈において、細胞療法は、心不全に向かって疾患の進行を停止させると、心筋の最近同定再生能を刺激するために新たな治療選択肢としてますます関心を集めている。実験的及び臨床的研究は、さまざまな細胞型の心臓移植後に得られる有益な効果の説得力のある証拠を提供した。主なアウトカムは改善されたカーディを含め交流収縮機能は、左心室リモデリングを減少させ、梗塞サイズを減少し、梗塞領域内の血管密度を増加させた。しかし、細胞注入後の低細胞数の保持は重要な欠点が残った。細胞送達2の改善するバイオマトリックスによる細胞の協会は最近、研究者や臨床関心を育んできた。 左冠動脈前下行枝（LAD）の連結は、経壁心筋梗塞および成熟瘢痕をもたらす小動物モデルにおける心筋梗塞のための参照方法です。 MIの慢性期に適用される細胞治療は、第二外科的介入を必要とします。胸骨正中切開は、通常、細胞またはbiograftsの心外膜注入の心筋内注入を可能にするために行われる。このような侵襲性外科手術手順は、死亡率、術後回復時間、痛み、感染症のリスクを高める。ここで紹介する低侵襲性のアプローチは、このようなバイアスを防ぐだけでなく、Oを提供するだけでなく治療適用のための心臓のptimalアクセス可能。 MIとゲル状のバイオマトリックスに関連する細胞の心外膜注入は左肋間開胸術を経由して拍動している心臓上で実行されます。

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:104px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">POLYSORB 3-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">POLYSORB 5-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SURGIPRO II 7-0 Suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">VP904X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CATHETER INSYTE 14G</td>
			<td style="width:104px;">BD</td>
			<td style="width:119px;">381267</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
			<td style="width:119px;">1501441</td>
			<td>TISSEEL Glue, Frozen -20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">72-8925</td>
			<td>use as retractor for the ribs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
			<td style="width:119px;">16044-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

心臓細胞療法は、細胞に関連生体材料の関心の高まりと注入が、心筋細胞送達を最適化するための大きな課題となって得ています。左前下動脈（LAD）結紮からなる心筋梗塞のげっ歯類モデル（MI）は、一般的に開胸を介して行われています。胸骨切開を介して第心臓切開手術は、伝統的に、治療の心外膜の用途のために行われている。 LAD結紮モデルの記述なので、手術後の死亡率は、しかし第二の手術が重要で推移している、百分の35から13まで低下した。術後の回復を改善し、痛みおよび感染を減少させるために、低侵襲性外科手術手順が提示される。二つの開胸術は、LAD結紮のための初期のものと治療心外膜投与のための第1を実施した。固体またはゲルタイプのマトリックスに関連した細胞からなるBiograftsは梗塞AR上に塗布し、EA。 2および6週後に行っ心エコー検査によって確認されるようにLAD結紮の心臓機能の喪失をもたらした。ゴールドナーは、心臓切片で実施トリクローム染色は、経壁瘢痕形成を確認した。第一及び第二の手術は少なく、その10％の術後死亡率をもたらした。

すべての動物は、開胸した後1時間以内に回復した。創傷治癒は急速であった。いいえ感染や浮腫が認められなかった。 左ダブル開胸、心臓（ 図1）への最適なアクセスを可能にした。痛みと手術後の死亡率は低かった。動物は、手術から素早く回収した（ 図2）体重が増えた。カプランメイヤー生存率は、最初開胸96％であった。 104以上の4匹の?...

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Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

げっ歯類モデルでLAD結紮により誘導される心筋梗塞を治療するためbiograftの移植は、従来、2つの心臓切開手術を必要としてきた。死亡率を減少させ、細胞に関連する、固体およびゼラチン状バイオマトリックスを固定するための最適な条件を提供するために、最小侵襲性処置が開発されてきた。

ラットにおける心不全のための細胞ベースの治療：心筋梗塞および細胞およびバイオマトリックスの心外膜移植のためにダブル開胸

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

cell-based-therapy-for-heart-failure-rat-double-thoracotomy-for

Research

JoVE Journal

Bioengineering

11.8K ビュー.  University of Fribourg. げっ歯類モデルでLAD結紮により誘導される心筋梗塞を治療するためbiograftの移植は、従来、2つの心臓切開手術を必要としてきた。死亡率を減少させ、細胞に関連する、固体およびゼラチン状バイオマトリックスを固定するための最適な条件を提供するために、最小侵襲性処置が開発されてきた。 

ビデオ: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

エンジニア心臓組織の移植後免疫反応の評価（EHT）のための生物発光イメージング

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

生物工学

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

心房と心室から心筋細胞における管状膜ネットワークの解析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

シードとマウス モデルにおける合成組織設計気管移植片の移植

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

ケラチン生成細胞増殖の 2 次元と 3 次元の評価タイプ I コラーゲン基板

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

磁気温熱療法とポト温熱併用がん治療のための磁気、音響、光三重応答性マイクロバブル

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

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

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

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

生体内心筋修復用ヒトiPS細胞由来のメッシュ状の設計心臓組織の調製

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

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心筋梗塞のマウスモデルにおける3次元バイオプリントパッチの移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

心臓血管磁気共鳴画像法によるマウス心左心室機能、心筋緊張、血行力の定量化

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2次元ヒト幹細胞由来心筋細胞における心収縮性調節療法の評価

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...