ソーニャSchrepfer、レナードコンラーディとアルネハンセンは、ドイツ学術振興（; SCHR992/3-1、SCHR992/4-1、CO 858/1-1、HA 3423/3-1 DFG）から資金提供を受けた。仕事はまた、欧州連合（EU）からトーマスEschenhagen（EUGeneHeartとAngioscaff）への補助金によって支えられている。

1。 EHTの生成と培養条件フィブリンベースのEHTの製造は、他の4に記載のように行った。簡単に言うと、移植の目的のためにフィブリンベースEHTを生成するために、マスターミックスは、新生児のROSA /ルシフェラーゼ- LEWトランスジェニックラットの心臓、ウシフィブリノーゲン及びマトリゲルから単離した細胞を含む調製した。アガロース鋳型は6ウェル培養皿に置かれたテフロンのスペーサを用いて調製した。アガロースの凝固した後、スペーサを除去し、シリコンポストラックは、鋳型に達するポストで培養皿上に置いた。 EHTを生成するには、マスターミックスは、簡単にトロンビンの計算量と混合し、金型に分注した。フィブリノーゲンの重合した後、ポストに付着EHTsシリコーンラックを静かにカスタムメイドを使用して、新しい6ウェル細胞培養皿に移し、37 ° C、7％CO 2インキュベーター内で維持さアガロースの鋳型、から削除されました細胞培養培地。 EHTは、日tenまで、細胞の培養条件下で飼育した。この期間中は、新生児ラットの心臓細胞は細長い、相互接続を開始し、シリコンの支柱の間に磁力線に沿って整列します。注入の時に、自発的な一貫性の収縮は、シリコーンの投稿を偏向、観察された。 2。 EHT注入 EHTの注入は、同種免疫応答を調べるために免疫ブラウンノルウェー（BN）ラット、または免疫不全ヌードラットのいずれかで行われた。 BNと100の重量を量るヌードラット - 150 gが、飼育標準ラット固形飼料および水を自由libidumをチャールズリバードイツ（Sandhoferウェグ7、D - 97633 Sulzfeld）から購入し、従来の条件下で飼育した。免疫不全ラットが使用されている無菌ベッド、ケージ、および滅菌水に収容されています。ドイツの倫理審査委員会は、日と動物の手順を承認した。すべての手術器具は使用前に滅菌されています。 移植手順は次のように実行されます。 ラットは、誘導チャンバーを用いて（2.5から3パーセント）イソフルランでanesthesizedされています。イソフルランに対するヒトの暴露量は、ダウンドラフトテーブルまたは非循環フードで働くことによって減らすことができます。体温は、手術中維持されます。 <ol><li>腹部領域のひげをそると麻酔中に乾燥から目を防ぐために、眼軟膏を適用する。 </li><li>その背中にネズミを置き、麻酔を維持するために、その鼻と口にフェイスマスクを置く。 </li><li> Betadineし、80％エタノールを用いて腹部を消毒する。坊主エリアの中心部からクリーンで汚れの綿棒は、髪の領域に向かって外側に移動。 </li><li>ラットは、十分に麻酔をかけていることを確認するために、後肢の足をつまんで反射神経をチェックしてください。 </li><li>動物をドレープし、腹部を開くには二つのステップで皮膚や筋肉を分離正中腹部切開を行います。ホットビーズの滅菌は、皮膚と筋肉の開口部との間で使用されています。 </li><li>暖めた生理食塩水しっとりパウダーフリー手袋で腸を置きます。水分の損失を防ぐために、腸の周りに手袋を折る。 </li><li>脂肪組織を削除し、脾臓を公開。 </li><li>鉗子で大網を視覚化して広げる。 </li><li>慎重に大網にシリコンのポストと、転送を中断からEHTをカット。 </li><li> EHTの周りに大きなomentusをラップと2つのシングル7から0 prolene縫合糸（エチコン、ノルダーシュテット、ドイツ）を使用して修正</li><li>次の腹部に戻して腸を動かす。 </li><li>温めておいた滅菌生理食塩水で腹部をフラッシュします。 </li><li> 6から0 prolene実行中の縫合糸（エチコン、ノルダーシュテット、ドイツ）を使用して腹壁の筋層を閉じます。 </li><li>皮膚を閉じるには、縫合糸を実行している5から0 prolene（エチコン、ノルダーシュテット、ドイツ）を使用してください。 </li><li>ラットが麻酔に入れたまま、皮下5 mg / kgのカルプロフェンを注入する。 </li><li>手順のこのタイプ、鎮痛が（そのようなMetamizolなど）3日後に移植のための鎮痛薬のために飲料水（100ml当たり50mgのMetamizol）に追加されるための十分な鎮痛を提供する。 </li></ol> 3。 EHT次の注入の生物発光イメージング IVISのバイオイメージングプラットフォームは（Xenogen、アラメダ、カリフォルニア州、米国）、in vivoで非侵襲的な生物発光イメージングに使用され、結果がIVISのリビングイメージ（Xenogen）ソフトウェアパッケージを用いて分析している。イメージングは​​、術後2時間を開始する毎日実行されます。 誘導チャンバーを用いて - （3％2.5）イソフルランでラットを麻酔。 <ol><li>広くプロボ-ヨウ素を使用して腹部と胸部領域を駆除する、次回の使用の80％エタノール、このステップを3回繰り返します。 </li><li> IP、D -ルシフェリン（375 mg / kg体重Xenogen）を注入</li><li> IVISイメージング室（最大4匹まで同時に解析することができる）に麻酔下のラットを置きます。 </li><li> Lの信号強度を評価するUC +光子/秒/ cm 2で / steridianinベースラインでの関心領域（ROI）（移植後120分）の単位で、日1、2、3、5、7、10時、および週2でEHT内部の細胞、 3と4。ピーク信号を（D -ルシフェリンの注射後20分の周りに表示）に注意してください。 </li></ol>時間をかけて信号強度を示すグラフ（Excel）を作成します。 4。 BLIの結果とELISPOTの相関免疫BNおよび免疫不全ヌードラットの細胞の生体内免疫応答は、EHT移植後に検討した。レシピエント動物の脾臓は、受信者の脾細胞を分離するために5日間EHT移植後に回収した。応答細胞がIFN -γとIL - 4のコーティングを使用して、製造元のプロトコル（BD Biosciences社）に従って行ったとして、ELISPOTアッセイを用いて刺激し、5 × 10 6受信者の脾臓細胞としてEHTsを（シグマアルドリッチ、セントルイス、MO、米国）マイトマイシンを抑制それぞれ、TH1 -、およびTH2応答を調査するプレート。 IFN -γ-及びIL - 4スポット免疫不全モデルに比べて免疫、モデルで有意に高かった（IFN -γ：P = 0.001、IL - 4：P = 0.023;スチューデントt -検定）。 5。代表的な結果： <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="図1" /> 図1。 BLIの結果とELISPOTの相関が ）免疫ブラウンノルウェーと免疫不全ヌードラットのin vivo免疫応答における細胞は、EHT移植後に検討した。受信者の脾細胞は、5日EHT注入後に回収した。 IFN？とIL - 4の発現は、応答細胞として刺激と5x106受信者の脾細胞としてマイトマイシン禁止EHTを使用してELISPOTアッセイにより評価した。 Th1およびTh2応答はそれぞれ、IFNγとIL - 4産生によって証明されるように免疫不全モデルに比べて免疫モデルで有意に高かった。 B）リュック+ EHTsは、免疫担当BNラットまたは免疫不全ヌードラットのいずれか大きいomentusに移植した。細胞の生存率は、縦方向にBLIが続いた。移植EHTsを拒否した動物の割合が表示されます。細胞はT細胞欠損ラットで生き残ったのに対し、すべてのBNラットは急速に、EHT移植を拒否。

<ol>
	<li>Eschenhagen, T., Zimmermann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+myocardial+tissue.">Engineering myocardial tissue.</a> Circ Res. 97, 1220-1231 (2005).</li><li>van der Bogt, K. E., Schrepfer, S., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transplantation+of+adipose+tissue-+and+bone+marrow-derived+mesenchymal+stem+cells+in+the+infarcted+heart.">Comparison of transplantation of adipose tissue- and bone marrow-derived mesenchymal stem cells in the infarcted heart.</a> Transplantation. 87, 642-652 (2009).</li><li>Hakamata, Y., Murakami, T., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Firefly+rats"+as+an+organ/cellular+source+for+long-term+in+vivo+bioluminescent+imaging.">"Firefly rats" as an organ/cellular source for long-term in vivo bioluminescent imaging.</a> Transplantation. 81, 1179-1184 (2006).</li><li>Hansen, A., Eder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Drug+Screening+Platform+Based+on+Engineered+Heart+Tissue.">Development of a Drug Screening Platform Based on Engineered Heart Tissue.</a> Circ Res. , (2010).</li></ol>

利害の衝突は宣言されません。

心臓修復のためのEHT技術の臨床実施の前に、そのような移植後の細胞とマトリックスの両方のコンポーネントの免疫原性や移植片生存としての基本的な質問は、実験モデルで評価する必要がある。 生体内生物発光イメージングは、移植後のモニタリングの細胞の生存のための有用な新技術を表します。 。我々は正常に確立プロトコル4の適応により、ルシフェラーゼ正（リュ?...

<ul><li> LEW - Tgは（GT（ROSA）26Sor - LUC）11Jmskラット（NBPRのラット番号0299、http://www.anim.med.kyoto-u.ac.jp/NBR/） </li><li>ウシフィブリノーゲン（ストック溶液：塩化ナトリウム200 mg / mlのプラスアプロチニン5μg/ mlのフィブリノゲン0.9％、シグマF4753） </li><li>マトリゲル（BDバイオサイエンス356235） </li><li> DMEM（生体色素F0415） </li><li>ウマ血清（ギブコ社26050） </li><li>ペニシリン/ストレプトマイシン（ギブコ15140） </li><li>インスリン（Sigma - Aldrich社I9278） </li><li>アプロチニン（シグマアルドリッチA1153） </li><li> D -ルシフェリン（Biosynth L - 8220） </li><li>酵素結合免疫スポット（ELISPOT）アッセイプレート（Hassa Laborbedarf、S2EM004M99） </li></ul>

bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (eht)

心臓組織工学の様々な技法がネイティブまたはバイオ人工どちら足場材料、などのフィブリンやコラーゲンなどのヒドロゲルにおける心筋細胞の捕捉と筋細胞の単層1のスタッキングを使用して足場の戦略などは過去数十年間で追求されています。これらの概念は、標的の心機能の回復（心筋梗塞後など）で、または実験的なモデル（例えば、予測毒性学と物質のスクリーニングや疾患のモデリング）として目指しています。人工心臓の組織の移植後の細胞生存率（EHT）の正確なモニタリングは、現在、生物発光イメージング（BLI）のテクニック2 -生体内で使用可能となった。ここでは、ホタルルシフェラーゼのユビキタス発現（、3 ROSA /ルシフェラーゼ- LEW Tg）を持つトランスジェニックラット系統からフィブリンベースのEHTの生成を説明します。注入はEHTの移植後の宿主の免疫応答を評価するために別のラット系統の大網に実行されます。 BLIと酵素結合免疫スポット法（ELISPOT）によって生成された結果の比較は、免疫応答の評価のためのBLIの有用性を確認する。

Watch this Scientific Journal Video about Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT at JoVE.com

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

このビデオはの使用方法を示しています<em&gt; in vivoで</emラットにおけるエンジニアの心臓組織（EHT）の移植後の免疫応答を研究する&gt;生物発光イメージング。

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 ...

bioluminescence-imaging-for-assessment-immune-responses-following

Research

JoVE Journal

Bioengineering

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

9.6K ビュー.  University Heart Center Hamburg. このビデオはの使用方法を示しています in vivoで生物発光イメージング。

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

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

ティッシュエンジニアーコンストラクトの成長評価のための磁気共鳴エラストグラフィの方法論

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

生物工学

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 ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

組織工学血管移植片とその後の移植のために手術手技<em&gt;インビボ</em&gt;モニタリング

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

偏神経組織のエンジニア3Dシルクコラーゲンベースモデル

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

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

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

心臓医薬品安全性スクリーニングのための人間エンジニア心臓組織の自動収縮分析

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

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

カスタム設計されたレーザー エッチングのマスターから培養金型

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

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 ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

ラットにおける円周食道再建のための組織工学型移植片

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

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 ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.