著者らは、培養するために図2に示す組織をYabin呉に感謝したいと思います、画像はバートバンOverbeekeによって撮影されました。仕事は財政的にSenterNovem、助成金、ISO 42022によってサポートされていました。

1。マウス筋芽細胞前駆細胞またはC2C12筋細胞の培養<ol><li>当初Sheferら10によって発行されて、後でCollins ら 11とBoonenはら 12によって適応プロトコルに従って細胞を分離し、液体窒素中でこれらを格納します。これは、C57BL / 6 例えば 、マウスを必要とします。代替方法は、 例えばLi Y らによって可視化実験誌に発表された方法では、他のラボで使用されています13。試薬や機器の入力は7ページと8ページの表を参照されます。 1マウスの筋からは、一般的に、液体窒素で約20バイアルを凍結保存するのに十分な細胞を得ることができます。次に、我々は、液体窒素貯蔵から細胞を解凍するためのプロトコルを起動します。 </li><li> 25cm 2のマトリゲル（1 mg / ml）でコーティングされた組織培養フラスコに1バイアルから細胞を入れて、から成る増殖培地（GM）（335ミリリットルDMEM高度、100ミリリットル胎児bovinを追加電子血清（FBS）、50ミリリットルのHS、5 mlのペニシリン/ストレプトマイシン、5ミリリットルのL-グルタミン、5mlのニワトリ胚エキス（CEE））。 </li></ol> 注：室温で2時間コートと吸引によってマトリゲルを削除します。 <ol start="3"><li> 3日ごとに細胞が約1:3サブカル。 これが意味する：3日目：1 75cm 2フラスコから2 150センチメートル2フラスコ（非コートフラスコでpreplating 30分付）への通路細胞：6日目に75cm 2フラスコに1 25cm 2のからの継代細胞。 1トリプル150センチメートル2フラスコに9日目の転送1 150センチメートル2フラスコに、または3 150センチメートル2フラスコ（必要に応じてpreplateが再び細胞の表現型に依存します）が利用できない場合。 12日目に細胞が筋肉構築物中に播種するための準備が整いました。 </li></ol> 注意： -トリプル150センチメートル2当たりの細胞数が約4.5×10 6個の細胞になります。 - を介して使用してください播種時の細胞の混合集団を得るためにそれらをミックスするための別のアイソレーションからのLS。 注：初代培養細胞で機能しないことを選択した場合、C2C12筋芽細胞は良い代替手段です。 2。エンジニアリング骨格筋組織<ol><li> &quot;硬化剤&quot;（10:1）を持つ &quot;エラストマー&quot;を混合することにより、シリコン接着剤を準備します。 1三角形の辺（; 図1形状の家のような）とマジックテープの5mm角のセグメント+ / -をカットします。正方形の間に約12mmのスペースで6ウェル培養プレートのウェルに接着剤ベルクロ。 </li></ol> 注： -のみベルクロのソフト面を使用し、この面を上に直面しています。 - 屋根がお互いに直面していることを確認してください。 - のみシリコン接着剤でマジックテープをカバーする、うまく全体に接着剤が広がらない。 - 後で電気刺激のためには、よくPLの垂直方向に構造物を揃えるために関連しています（長軸に沿って）食べました。 <ol start="2"><li>気泡を除去するために主に、加熱されていない、真空オーブンで一晩乾燥させるままにしておきます。 15分間インキュベートしたウェルに70％エタノールを添加することにより滅菌する。 PBSで3回リンスし、15分間UV下で置く。ウェルと使用するまでインキュベーター内ベルクロと場所からすべてのPBSを除去。 </li><li>冷蔵庫にマトリゲルソリューションを解凍し、まもなく3D構造物を作る前に所望の濃度にコラーゲン溶液を調製します。無菌の0.02パーセント酢酸（最終濃度3.2 mg / ml）を持つ株式コラーゲンを希釈します。 </li></ol> 注：氷の上のすべてのものを残す。 <ol start="4"><li> GMとカウントに再懸濁した細胞を、トリプシン処理。インキュベーター内で遠心分離チューブに細胞を残す。 </li><li>淡いピンクのcoloまで、このコラーゲン溶液に0.5M NaOHを加えるには、チューブ（ 表1によると）にしたい構造物の番号については、コラーゲン原液の所望量を追加rは7.5のpHを示す情報である。とを上下にピペッティングにより穏やかに混合し、バブルが形成されるのを防ぐ。その後、マトリゲルを追加し、優しく、しかし、非常に徹底的に混ぜる。最後に、コラーゲン/マトリゲル混合物（適切な量については表1を参照）にGMを追加します。 </li></ol> 注意： -氷の上に各ステップを実行します！マトリゲルおよびコラーゲンは温度の上昇で容易にゲルだろう。 - 色は確かにピンクであることを覚えて！ pHの上昇はまた急激なゲル化を誘導する。 - コラーゲンの最終濃度：1.6 mg / mlである。 <ol start="6"><li> 5分間1,000 rpmで細胞の所望量を遠心し、上清を除去する。 </li></ol> 注 -構築あたりの細胞数を調整する必要がある細胞の活性に応じて。一般的に、細胞数は、構築物75万の間と125万細胞に位置しています。 <ol start="7"><li>細胞ペレットを再懸濁するためにゲル混合物の一部を使用そして、残りの混合物中に細胞を移し、よく混ぜますが、気泡を導入することなく。 </li><li>インキュベーターとピペットベルクロ第一に、セルゲル混合物の0.35 -0.4 mlの予熱ウェルプレートを取り出します。次に、結合部位間の中心から混合物をピペットとベルクロに拡張し始める。最後に、ベルクロ部分の周りに2つのベルクロアンカーとピペットとの間のギャップを埋めるためにゲルの残りの部分を使用しています。 </li><li>ゲルが転送されるのに十分な固体であれば慎重に5〜10分後にチェック、 すなわち優しく料理をタップして、ゲルが剛体であれば視覚的に検査する。もしそうであれば、慎重にインキュベーターに皿を置きます。通常、それらは10分後にピックアップすることができます。その後、1〜2時間後に、優しく温かみのあるGMの4mlで各ゲルを重ねます。 </li></ol> 注：-DOプレートを取り扱う際には、任意の活発な動きをしない。 <ol start="10"><li> （増殖培地分化培地でGMを置き換える24時間後）ニワトリ胚エキスを伴うものではなく、2〜3日毎に新鮮な培地と交換してください。 7日目には、溶融筋線維に横紋開発のための染色を評価することができるように、成熟した指向の筋繊維を取得している必要があります。 </li></ol> 3。電気的な刺激<ol><li>安全キャビネット内の紫外線の下で、70％エタノール、続いて乾いた、使用前（試薬や機器の表を参照）ionoptixプレートから電極を滅菌する。コンストラクトを培養プレート上に電極とプレートを置き、培養プレートインキュベーターへの転送から蓋で覆う。適切なケーブルを使用して、C-Ionoptixペーサーを接続します。 </li></ol> 注：電極は筋肉コンストラクト（ 図2）と一緒に平行に配置されている。 <ol start="2"><li>予想通り刺激プロトコルを適用します。 </li></ol> 注：我々は、一般的に、4 V / CMを使用する2Hzの周波数で6msのパルス。刺激中に培地ごとに24時間を変更します。

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著者らは、開示することは何もない。

筋肉組織のエンジニアリングは、疾患モデルとして、薬剤スクリーニングのために、再生医療及び食肉生産のために使用するための大きな可能性を秘めている。ただし、これらのアプリケーションのための要件は異なります。コラーゲンは、細胞整列を可能にするため、および以前の2Dの研究12で決定された筋芽細胞の前駆細胞が基底膜由来タンパク質の存在を必要とするため、我々?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>マトリゲル-成長因子低減</td><td>ベクトン·ディッキンソンと</td><td></td><td></td></tr><tr><td> DMEM（高グルコース）* </td><td>ギブコ</td><td> 42430 </td><td></td></tr><tr><td>アドバンストDMEM </td><td>ギブコ</td><td> 12491 </td><td></td></tr><tr><td>ウマ血清</td><td>ギブコ</td><td> 65050-122 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>グライナー</td><td> 758075 </td><td></td></tr><tr><td> 0.45及び0.22μmのシリンジフィルター* </td><td>ワットマン（シュライヒャーとScheull） </td><td> 10462100 </td><td></td></tr><tr><td> L-グルタミン酸</td><td>ギブコ</td><td> 25030024 </td><td></td></tr><tR&gt; <td>ペニシリン/ストレプトマイシン</td><td>ギブコ</td><td> 10378016 </td><td></td></tr><tr><td>アンフォテリシン</td><td>ギブコ</td><td> 15290-018 </td><td></td></tr><tr><td>培養プラスチック</td><td>グライナー</td><td></td><td>培養フラスコとピペットを含む</td></tr><tr><td>ニワトリ胚エキス</td><td>米国生物</td><td> C3500から</td><td></td></tr><tr><td>パスツールピペット* </td><td> Hilgenberg </td><td></td><td> - 1,1ミリメートル0,9の先端径は230ミリメートル：綿、開口先端Lとくびれのあるパスツールピペット、、 </td></tr><tr><td>パスツールピペット* </td><td> Hilgenberg </td><td></td><td>綿、開口先端Lとくびれとパスツールピペット、、：1,4の先端直径が230ミリメートル - 1.6ミリメートル</td></tr><tr><td>パスツールピペット</td><td> VWR </td><td> 612-1702 </td><td></td></tr><tr><td>コラゲナーゼI型* </td><td>シグマ</td><td> C0130-16 </td><td></td></tr><tr><td> 40μmのセルストレーナー* </td><td> BDファルコン</td><td> 352340 </td><td></td></tr><tr><td> 19G針</td><td></td><td></td><td></td></tr><tr><td>エラストマー</td><td>ダウコーニングコーポレーション</td><td> 3097358-1004 </td><td>シラスティックMDXの＃4から4210 </td></tr><tr><td>硬化剤</td><td>ダウコーニングコーポレーション</td><td></td><td>シラスティックMDXの＃4から4210 </td></tr><tr><td>ベルクロ</td><td>正規店</td><td></td><td>あなただけのソフト面を使用し、正規の店でこれを購入することができます</td></tr><tr><td> I型コラーゲン、ラットの尾</td><td> BD Biosciences社</td><td> 3544236 </td><td></td></tr><tr><td> C-PACE EP文化ペーサー</td><td> Ionoptix </td><td></td><td></td></tr><tr><td>電気刺激のための6ウェル培養皿</td><td>ベックktonディッキンソン·ファルコン</td><td> BDファルコン＃353846 </td><td></td></tr><tr><td> C-ディッシュ培養皿電極</td><td> Ionoptix </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> *細胞の単離（1.1ポイント）のために必要 ＃一緒に一つのキットで</td></tr></tbody></table>

engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation

仕組まれた筋肉組織は、褥瘡を勉強するなど、in vitroでの疾患モデルとして、再生医療用や肉代替の1として使用するための組織の生産を含む、いくつかの異なる目的に使用できます。最初に報告された3D筋肉構築物は、何年も前に作られ、分野の先駆者はVandenburghや同僚2,3アールされています。筋肉組織工学の進歩は、生化学的要因、幹細胞や前駆細胞の知識の広大なゲインから結果だけではありませんが、特に、物理的な要因が細胞挙動の制御において重要な役割を果たし、その研究によって得られた洞察に基づいている組織開発。最先端の人工筋肉は、現在セル人口ヒドロゲル構造物で構成されて構築されます。私たちの研究室では、これらは一般的に、C2C12、マイルマウス後肢の筋肉から単離されたマウス筋芽細胞の前駆細胞、又はマウス筋芽細胞株で構成されコラーゲン/マトリゲルの混合物でXEDと2アンカーポイントの間で播種し、筋肉の靭帯を模倣する。他のセルは、同様にそのようなL6ラット筋芽細胞4、新生児筋由来前駆細胞は5、そのような人間の6あるいは誘導多能性幹細胞（iPS細胞）7などの他の種由来の成体筋組織由来の細胞として例えば代替細胞株を考慮することができる。細胞収縮は、構築物8,9と文化の約1週間後の筋前駆細胞の分化の長軸に沿った細胞のアラインメントを引き起こします。また、電気刺激の適用はある程度8への分化のプロセスを向上させることができます。その限られたサイズ（8×2×0.5ミリメートル）のために完全な組織は、 例えば生存、分化、細胞アライメントを監視するために、共焦点顕微鏡を用いて分析することができる。特定のアプリケーションにengineeの要件に応じて赤い筋肉組織は変化するであろう、他の種への肉の代替の翻訳が必要であるとして機能するようにしながら再生医療用など使用は、組織の大きさと血管新生のアップスケーリングが必要になります。

最終製品は、 図3に示されている筋肉の構成要素となります。組織の大きさは長さ約8ミリメートル、幅2mm、厚さ0.5mmとなります。分化中の電気刺激は、ミオシン重鎖アイソフォームの発現を変更しますが、分化培地8によって誘導されるように大きく分化過程を強化しませんが、電気刺激は、筋肉の機能性をチェックするために、プロセスの終了時に適用することができますなぜ?...

Watch this Scientific Journal Video about Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation at JoVE.com

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

仕組まれた筋肉組織は、疾患モデルとして、また肉の代替源として、再生医療における大きな可能性を秘めている。ここでは、マウス筋芽細胞の前駆細胞、及び電気パルスによる刺激からこのケースでは、筋肉の構造物の工学を記述します。

電気刺激のマウス筋芽細胞前駆細胞およびアプリケーションからの工学骨格筋組織

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

engineering-skeletal-muscle-tissues-from-murine-myoblast-progenitor

Research

JoVE Journal

Bioengineering

20.9K ビュー.  Eindhoven University of Technology, The Netherlands. 仕組まれた筋肉組織は、疾患モデルとして、また肉の代替源として、再生医療における大きな可能性を秘めている。ここでは、マウス筋芽細胞の前駆細胞、及び電気パルスによる刺激からこのケースでは、筋肉の構造物の工学を記述します。

ビデオ: Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

コラーゲンゲル含有する細胞のモジュールを使用してマイクロ組織の作製

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

生物工学

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

エンジニアリングフィブリンベースの組織の筋線維芽細胞と制約の適用からの構築と細胞とコラーゲン（再）組織を誘導するためにひずみ

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

マウス胚性幹細胞からの神経組織エンジニアリングのためのポリエレクトロ繊維状足場（グリセロール - ドデカン）

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

心筋細胞ベースのアクチュエータおよび自己安定化バイオロゴ - パート1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. These devices, called biorobots, are powered solely by the force generated from the contractile activity of the living component and, due to their many inherent advantages, could be an alternative to conventional fully artificial robots. Here, we describe the methods to seed and characterize a biological actuator and a biorobot that was designed, fabricated, and functionalized in the first part of this two-part article. Fabricated biological actuator and biorobot devices composed of a polydimethylsiloxane (PDMS) base and a thin film cantilever were functionalized for cell attachment with fibronectin. Following functionalization, neonatal rat cardiomyocytes were seeded onto the PDMS cantilever arm at a high density, resulting in a confluent cell sheet. The devices were imaged every day and the movement of the cantilever arms was analyzed. On the second day after seeding, we observed the bending of the cantilever arms due to the forces exerted by the cells during spontaneous contractions. Upon quantitative analysis of the cantilever bending, a gradual increase in the surface stress exerted by the cells as they matured over time was observed. Likewise, we observed movement of the biorobot due to the actuation of the PDMS cantilever arm, which acted as a fin. Upon quantification of the swimming profiles of the devices, various propulsion modes were observed, which were influenced by the resting angle of the fin. The direction of motion and the beating frequency were also determined by the resting angle of the fin, and a maximum swim velocity of 142 &#181;m/s was observed. In this manuscript, we describe the procedure for populating the fabricated devices with cardiomyocytes, as well as for the assessment of the biological actuator and biorobot activity.

In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time.

心筋細胞ベースのアクチュエータと自己安定化ビオロボ - 第2部

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

マイクロ流体工学バイオプリンティング血管組織や Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

組織工学用植物組織の Decellularization の 2 つの方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

細胞の Cardiomyogenic の可能性を高めるに同時電気的・機械的刺激

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

建設と質間葉系幹細胞の骨分化を強化するため電気刺激室の使用

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.