トレイシーC. Grikscheit、エリック·R·バーセル、フレデリック·G·サラは、カリフォルニア再生医療研究所（CIRM）、助成番号RN2-00946から1（TCG）とTG2-01168（ERB、FGS）によってサポートされています。アリソン·L·シュペーアは大学外科エチコン学者の協会です。 Yashuhiro Torashimaは小児病院ロサンゼルスサバン研究所研究キャリア開発奨学金によって賄われています。

1。オルガノイドユニットの準備<ol><li>マウスの解剖（はさみや鉗子）のための適切なインスツルは、オートクレーブにより滅菌しなければならない。 </li><li>人道的に地元の動物実験委員会のプロトコルに従ってドナーマウスを安楽死させる。動物が進む前に死んでいることを確認してください。 </li><li>腹膜腔へのアクセスを得るために正中切開を行います。露出を改善するために必要なスキンフラップは反映させることができます。 </li><li>小腸を骨抜きとトライツ靱帯のすぐ遠位で割ります。シャープで優しい鈍的切開を使用して、その腸間膜から小腸を分離します。回盲部を識別し、これに近位小腸5ミリメートルを分割します。 </li><li>はさみを使用して、（インビトロジェン、アンチアンチ）10ミリリットル4℃、無菌ハンクス緩衝生理食塩水（HBSS、インビトロジェン、カールスバッド、カリフォルニア州）/ 1X抗生物質抗真菌薬でシャーレに腸間膜反対側縁に沿って長手方向にソリューションを腸を開きます。で開かれ、腸から糞便をクリア穏やかに撹拌し、次に4の10ミリリットル℃、滅菌HBSS / 1X抗抗15ミリリットル遠心チューブに開いた小腸を転送します。 </li><li> 4の10ミリリットル℃、試験管内抗抗滅菌HBSS / 1Xで開か腸を3回洗浄します。各洗浄は30秒間、15ミリリットル管の軽度の揺れを用いて行うことができる。振とう後、腸組織はチューブの底に沈む。間葉系デブリである浮遊物質を、捨ててください。ピペットで慎重に洗浄溶液を除去します。 </li><li> 4の10ミリリットル℃、はさみを使用して1mm未満正方形片に抗抗滅菌HBSS / 1Xとペトリ皿の中で洗浄した腸にミンチ。自動ピペットでミンチ材料を集め、試験管に入れます。 </li><li> 8分間500rpmでチューブを遠心分離します。脂肪と間充織含む上清を捨てる。 </li><li>滅菌HBSS / 1X抗抗プラス0.125 mg / mlのディスパーゼ（Invitrogen）および800単位/ mの10mlでみじん切り、洗浄した物質を消化リットルコラゲナーゼタイプ1（ワージントン、レイクウッドニュージャージー州）。消化溶液40mlを準備するには、ディスパーゼ、コラゲナーゼの142 mgを5 mgを秤量し、40mlの体積に滅菌HBSSを加算します。新たに各時間オルガノイドユニットが用意されているこの溶液を調製し、使い4℃まで、準備完了状態に保つ。ステップ1.8からペレットに直接消化溶液を追加します。 </li><li> 20分間37℃で消化液でみじん切り、洗浄材料を含有する試験管をインキュベートする。 </li><li>試験管を取り出し、さらに10mlのピペットを用いて磨砕することによって消化された組織を混乱させる。均一な外観が得られるまで、20から50回の間で繰り返します。 </li><li> 800 rpmで5分間試験管を遠心分離します。単一細胞を含む上清を捨てる。 </li><li> 4℃、滅菌したダルベッコ改変イーグル培地（DMEM、Invitrogen）にプラス10％（v / v）の熱不活化ウシ胎児血清（HI-FBS、Invitrogen）を10mlの消化反応を停止します。ペレを再懸濁しトンとチューブを横に振る。 </li><li> 800 rpmで5分間試験管を遠心分離します。最後の数滴まで、自動ピペットで注意深く上清を取り除きます。ペレットを再懸濁し避けるために最後の数滴のための使い捨てのプラスチックピペットを使用しています。 </li></ol> 2。ポリグリコール酸の足場のロード<ol><li>不織布、ポリグリコール酸（2mmの板厚、60mgのcm-3の嵩密度、気孔率&gt; 95％、コンコルディア繊維、コベントリー、RI）からフォームが2 mm長、5 mmの外径が円筒形の足場文献に記載されているよう。 4。 </li><li>蒸留水に70％エタノールを用いて調製し、ハサミで転倒使い捨て千マイクロピペットの先端2mmをトリムします。 </li><li> 4ウェル培養プレートに足場を置きます。第1のルーメンにしてから、外側の表面に、1000マイクロリットルピペットで足場上オルガノイドユニットをロード。内腔の被覆を確実にするためにピンセットを使用しています。妨害したり、ポリマーの円筒形状を開いて壊してはいけません。 </LI&gt; </ol> 3。ホストマウスへの移植<ol><li>可能な場合はドナーと同じ背景に同系宿主マウスを使用してください。それ以外の場合は、（ジャクソン研究所、サクラメントカリフォルニア州）、免疫不全非肥満糖尿病/重症複合免疫不全またはNOD / SCID動物を採用しています。 </li><li>イソフルランで全身麻酔を誘導する。 、prepを剃るとマウスの腹部をドレープ。 </li><li>腹膜腔への入り口を得るために5ミリメートル正中切開を行います。識別し、慎重に大網を骨抜きにする。大網上にロードされたポリマーを置き、ティッシュで包んでください。大網が破れないようにしてください。 </li><li> 5から0 monocryl縫合糸で大網にポリマーを固定します。そっとその解剖学的位置に包まれたポリマーと大網を交換してください。 </li><li> 4から0ビクリル縫合糸を使用して、レイヤーで腹部切開を閉じます。筋肉の閉鎖を実行し、切開下腹部内臓を傷つけないように注意してください。肌のために中断された縫合糸を使用して、。 </li><li>切開に隣接皮下膨疹として滅菌水で2 mg / kgのケトプロフェン（Ketofen、フォートダッジ·アニマルヘルス）による術後鎮痛を管理します。動物を毎日評価されるべきであり、動物が痛みや苦痛の徴候を実証されている場合は、ケトプロフェンの追加投与は術後2日目に投与してもよい。術後3日目までに、動物は完全に痛みや苦痛の証拠なしで回復されるべきである。痛みや苦痛は、術後3日目に続く場合は、これは異常とみなされ、動物実験委員会及び動物飼育施設のプロトコールに従って対処すべきである。 </li><li>静養するには、マウスや組織工学腸が4週間にわたって成長させる。 Septra 200ミリグラム/ 1:100希釈で5ミリリットル当たり40ミリグラム（ハイテクPharmacal、アミティニューヨーク州）とげっ歯類（ラボダイエット5001、PMIニュートリション、セントルイス、ミズーリ州）と水に動物の不断のアクセス権を与えます。 </li></ol> 4。収穫<ol><li>フーマーnely 4週間注入した後、宿主動物を安楽死させる。 </li><li>元の切開をもう一度開いて、腹膜腔へのアクセスを容易にするために頭の方にスキンが反映されています。 </li><li>筋層を開き、組織の地球儀として組織工学構築物を識別します。 </li><li>鋭い解剖を用いて腹腔内内臓からコンストラクトに癒着を降ろす。 </li><li>取り付け後パラフィンホルマリンでコンストラクトを修正するか、そのようなリアルタイムPCRまたはタンパク質の単離などの生化学的アッセイのための新鮮な組織を使用しています。 </li></ol>

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Surg. 243, 756-766 (2006).</li><li>Reyes, J., Bueno, J., Kocoshis, 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=Current+status+of+intestinal+transplantation+in+children.">Current status of intestinal transplantation in children.</a> J. Ped. Surg. 33, 243-254 (1998).</li></ol>

特別な利害関係は宣言されません。

我々はオルガノイドユニットオン足場のアプローチを用いて、マウスにおける組織工学腸を製造するためのプロトコルを提示。最も重要なステップは、オルガノイドユニットの準備のものです。ケアを十分に清掃し、機械的に組織を処理するために注意しなければなりませんが、消化が行われた後、平等なケアを行う（ステップ1.11）オルガノイドユニットをover​​digestまたはovertriturateない?...

<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> HBSS </td><td>ギブコ</td><td> 114170-112 </td><td></td></tr><tr><td>抗生物質抗真菌100X </td><td>インビトロジェン</td><td> 15240-062 </td><td></td></tr><tr><td>ディスパーゼ</td><td>ギブコ</td><td> 17105-041 </td><td></td></tr><tr><td>コラゲナーゼタイプ1 </td><td>ワージントン</td><td> LS004194 </td><td></td></tr><tr><td> DMEM高グルコース1X </td><td>ギブコ</td><td> 11995-065 </td><td></td></tr><tr><td>熱不活性化FBS </td><td>インビトロジェン</td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt 100パーセントのPGA </td><td>コンコルディアメディカル</td><td> FELT01-1005 </td><td>献などのポリマーを調製するための。4 </td></tr><tr><td>ポリ-L-乳酸</td><td> Durect </td><td> B6002-1 </td><td>献などのポリマーを調製するための。 4 </td></tr><tr><td> I型コラーゲン、ラットの尾を入力</td><td>シグマアルドリッチ</td><td> C3867-1VL </td><td>献などのポリマーを調製するための。 4 </td></tr><tr><td>ケトプロフェン100 mg / mlの</td><td>フォートダッジ·アニマルヘルス</td><td> 71-KETOI-100から50 </td><td></td></tr><tr><td> LabDiet 5001げっ歯類</td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> Septra 200ミリグラム/ 5ミリリットル、USPあたり40ミリグラム</td><td>ハイテクPharmacal </td><td> 50383-824-16 </td><td></td></tr><tr><td>イソフルランは、USP </td><td>フェニックス医薬品</td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

組織工学小腸（TESI）が正常に40日以内に術前の重みに戻り、その結果、大量の小腸切除後のルイスラットを救出するために使用されてきました。ヒトでは1、巨大小腸切除は、短腸症候群になる可能性があり、機能的な吸収不良非経口栄養依存性、肝不全や肝硬変、multivisceral臓器移植の必要性を含む重要な罹患率、死亡率、医療費を付与する。2本論文では状態、我々はマウスモデルにおける組織工学腸を作成するための我々のプロトコルを記述し、文書化多細胞オルガノイドユニットオン足場アプローチ。オルガノイドユニットは粘膜と間葉の両方の要素を含む腸由来の多細胞集合体、3腸管幹細胞ニッチを維持している間に関係があります。進行中および今後の研究では4日に私たちの技術の変遷マウスは、この種で利用可能なトランスジェニックのツールを利用することによってTESI形成の間に関与するプロセスの調査が可能になります。免疫不全マウス系統の5可用性はまた、私たちは人間の腸組織への技術を適用し、人間のTESIの形成を最適化することを許可しますヒトへの移行前にマウス異種移植。我々の手法は、すでに人間の患者での使用が承認された適正製造基準（GMP）の試薬および材料を採用していますので、脱細胞化された動物の組織に頼るのアプローチに比べて大きな利点を提供しています。このメソッドの究極の目標は、短腸症候群に対する再生医療の治療戦略としてのヒトへの翻訳である。

図1は、ここに記載されたプロトコルの全体的なスキーマを示しています。このプロトコルの最終的な結果は、地球やルーメン、粘膜、粘膜下組織と組織工学マウス小腸の球状の構造になっており、筋図2Aを囲むと、原料ポリマー足場と比較して、典型的な地球を、 図2（b）は、同じ構造を表示します急激にその内腔を明らかにするためにbivalved 図3...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

この記事とそれに付随するビデオはオルガノイドユニットオン足場のアプローチを使用して、マウスにおける組織工学腸を生成するための我々のプロトコルを提示します。

マウスモデルにおける腸のティッシュエンジニアリング

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

tissue-engineering-of-the-intestine-in-a-murine-model

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JoVE Journal

Bioengineering

14.8K ビュー.  Keck School of Medicine of the University of Southern California. この記事とそれに付随するビデオはオルガノイドユニットオン足場のアプローチを使用して、マウスにおける組織工学腸を生成するための我々のプロトコルを提示します。

ビデオ: Tissue Engineering of the Intestine in a Murine Model

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 ...

生物工学

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

組織工学のためのエレクトロ繊維のポスト処理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

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

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 study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

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 ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

組織工学のための二軸メカニカルローディングバイオリアクターの設計

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

中に組み込み血管新生による組織工学<em&gt;インビボ</em&gt;ティッシュ・エンジニアリング商工会議所

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

血管新生および血管組織工学を研究するための小さな動物モデルにおける動静脈（AV）ループ

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

運動調節血清による靭帯構築物の治療：翻訳組織工学モデル

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...