我々は、資金調達のために米国心臓協会中部大西洋のアフィリエイトを（0555407U、GLB）に感謝いたします。

<ol><li>エレクトロスピニングする前に、コラーゲンは、6ヶ月のウシ真皮から、酢酸ベースのプロセスを使用して抽出する必要があります。この抽出されたコラーゲンは、その後解散、沈殿、およびdialysisの複数形のそれに続く一連の精製される。一度コラーゲンを凍結乾燥され、エレクトロスピニングプロセスを開始できます。 </li><li>ポリカプロラクトン（PCL）、エラスチン（ELAS）、及びコラーゲン（COL）：3つの材料がたちこめている。これらの材料は別々にそれぞれ100 mg / mlの、200 mg / mlの、および70 mg / mlの濃度で1,1,1,3,3,3ヘキサフルオロ-2 - プロパノール（HFP、TCIアメリカ）に溶解されます。 </li><li>材料は98-2-0 PCL - ELAS - COL（内膜）、45-45-10、55-35-10、および65-25-10 PCL - ELAS - COLの体積比が少なくとも5つの異なるシンチレーションバイアル中でブレンドされています（メディア）、および70-0-30 PCL - ELAS - COL（外膜）。 </li><li>バースト、縫合糸の保持、およびコンプライアンステストのための多層人工血管を3〜1構成からエレクトロスピニングされたまま一軸引張試験および個々の層のコンプライアンステストに、材料のそれぞれは、個別にエレクトロだった。一軸引張試験の足場が平坦で長方形であった（2.5 cm幅X 10.2 cm厚× 0.3 cmの長さ）個々の層と多層の移植をelectrospinするために使用される直径2mmのマンドレルからは、試験試料を打ち抜きのために余りに小さかった。 </li><li>個々とマルチレイヤの両方エレクトロスピニングの場合は、ポリマー溶液は、18ゲージ鈍先端針付きプラスチック3ミリリットルBecton Dickinson社製の注射器に充填した、と一連の診療所の速度でシリンジポンプの上に置く。多層エレクトロレートは転移と層に応じて変化している間、個々のエレクトロスピニングのレートは、4ミリリットル/時間に設定されていました。流量とボリュームは、このような通りであった：内膜は4ミリリットル/時間の速度でエレクトロであり、0.5mlの容積は2ml /時間ごとに0.2 mlの内膜と中膜注射器の両方を組み合わせて、遷移が続きます。内膜注射器は、遮断され、内側層を2ml /それぞれの時間ではポリマーの溶液0.2mlに対して、メディアと外膜の間の遷移に続いて4ミリリットル/ hrで0.6ミリリットルのためにスピンさせた。最後に、メディアを停止し、外膜を4ml /時で0.4 mlのスピンさせた。 </li><li>すべてのサンプルは前にどんなテスト1〜18時間、50 mMのEDCで架橋された。 </li><li>一軸引張試験は、24時間PBSで水和oneエレクトロスピニングシートから6つのサンプルで実施した。 &quot;ドッグボーン&quot;型のサンプルは、エレクトロマットから打ち抜き、100 Nのロードセルと10.0 mm /分の延長率とのMTS Bionix 200のテストシステムでテストされました。ピーク応力、弾性率、および破断時のひずみはTestWorksのバージョン4を用いて計算した。 </li><li>縫合糸保持テストは、50 Nのロードセル（MTS Systems社）とMTS Bionix 200テストシステム上で37℃で24時間PBSに浸し、6種類のエレクトログラフト、° C、より6枚の内径管状試験片を実施したと米国規格協会2で説明した手順でストレートに基づき150.0ミリメートル/分の延長率。 5から0までの商業PDS II紫のモノフィラメント縫合糸は、試料の端から2 mmを配置し、縫合糸は、移植片を通して引っ張っていたまで延長された。ピーク負荷はTestWorksのバージョン4を使用してグラム - 力で記録された。 </li><li>破裂強度試験は、6つの異なるエレクトロ移植から6つのサンプルで実施した。バーストチューブ、長さ2-3 cmは、デバイスに接続されている、と2から0絹縫合糸で固定さ1.5ミリメートル直径の乳首に装着、PBSで水和した。空気は、チューブは2バーストまでの5 mmHgの/ sの速度で圧力を増加させる、システムに導入されました。結果は、チューブが破裂したときのmmHgの中の圧力として報告されます。 </li><li>ダイナミックコンプライアンスは、シミュレートされた生理的条件下で3cmの長さで6種類のエレクトログラフトから取られた6枚の内径管状移植のために決定した。個々の層と多層のチューブ状構造の両方が、同じ条件下でエレクトロスピニングとテストされました。試料は37℃のPBSで満たした組織成長の技術により開発された機械的な刺激（ITEMS）バイオリアクター℃に介してインテリジェントティッシュエンジニアリングで試験したバイオリアクターは、移植片の内側に、50分の90の3つの異なる圧力レベル、80分の120に1Hzの周期的な圧力変化を提供し、110分の150 mmHgの収縮期/拡張期2を検討した、3。 </li></ol>代表的な結果エレクトロスピニングのプロトコルが正しく行われている場合、最終製品は、ソフト、シームレス管の層の間に剥離のない初期の兆候としてください。一軸引張試験、破裂強度試験、縫合糸の保持テスト、およびコンプライアンスのテストが実行されると、結果は、内側層の剛性が増大するにつれて、エラスチンの減少量と、関連する機械的性質は、硬めのチューブを示す必要があることを示す必要があります。

<ol>
	<li>McClure, M. J., Sell, S. A., Barnes, C. P., Bowen, W. C., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+Electrospun+Polydioxanone-Soluble+Elastin+Blends:+Material+Characterization.">Cross-linking Electrospun Polydioxanone-Soluble Elastin Blends: Material Characterization.</a> Journal of Engineered Fibers and Fabrics. 3, 1-10 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiovascular implants - Vascular graft protheses. VP20, (1994).</li><li>McClure, M. J., Sell, S. A., Simpson, D. G., Walpoth, B. H., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-layered+electrospun+matrix+to+mimic+native+arterial+architecture+using+polycaprolactone,+elastin,+and+collagen:+A+preliminary+study.">A three-layered electrospun matrix to mimic native arterial architecture using polycaprolactone, elastin, and collagen: A preliminary study.</a> Acta Biomater. , (2010).</li></ol>

何人かの著者は、米国および本稿で提示に関する技術を保留中の国際特許を持っている、そしてこの技術は、器官形成、（株）にライセンス供与されている

この研究の最も重要な部分は、エレクトロスピニングプロセスです。 3月1日入力 - 出力のノズルを使用する場合は、アーチ形のと電荷損失が発生する可能性があります。これが発生した場合、回転しているポリマーに関連付けられている電圧は溶接、&quot;ウェット&quot;繊維を引き起こすと三層のそれぞれの間の剥離を作成し減少します。したがって、一貫性の電位は、理想的な多層チューブ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Polycaprolactone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Elastin</td>
<td>&nbsp;</td>
<td>Elastin Products Company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Acid</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TRIS</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 L Avanti J-HC Centrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1,1,1,3,3,3 hexafluoro-2-propanol</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3 ml Becton Dickinson Syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CZE1000R Rack Mount Power Supply</td>
<td>&nbsp;</td>
<td>Spellman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>High-Pressure Syringe Pump</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>70 % ethanol</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ImageTool 3.0 software</td>
<td>&nbsp;</td>
<td>Shareware provided by UTHSCSA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scanning Electron Microscope</td>
<td>&nbsp;</td>
<td>Zeiss EV050</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MTS Bionix 200 testing system</td>
<td>&nbsp;</td>
<td>MTS Systems Corp.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TestWorks version 4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Intelligent Tissue Engineering via Mechanical Stimulation (ITEMS) Bioreactor</td>
<td>&nbsp;</td>
<td>Tissue Growth Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

tri-layered electrospinning to mimic native arterial architecture using polycaprolactone, elastin, and collagen: a preliminary study

ネイティブ動脈を通じて、コラーゲンとエラスチンは、機械的なバックボーンを提供する血管の破裂を防止し、拍動性変形下での回復を促進する、重要な役割を果たす。本研究の目的は、45-45-10のブレンド、55から35とエラスチンとコラーゲンの添加によりポリ（カプロラクトン）（PCL）で構成される多層エレクトロ導管を作製することによって、ネイティブ動脈の構造を模倣することでした - 10、65-25-10 PCL - ELAS - COL組織再生に資する維持しながら、ネイティブ動脈組織を示す機械的性質を実証する。全体の移植と個々の層は、一軸引張試験、動的コンプライアンス、縫合糸の保持、および破裂強度を用いて分析した。 11.8 MPaの - 一軸結果が2.0の平均弾性率の範囲を明らかにしながら2.8パーセント/ 100 mmHgの、 - コンプライアンスの結果は、内側/中間層への変更は、0.8に至るまで全体の移植片のコンプライアンス値を持つ全体的な移植片の動作を変更したことを明らかにした。係数とコンプライアンスデータの両方がネイティブ動脈の範囲内の値を表示します。数学的モデリングは、層の剛性の変化が全体の円周方向壁応力をどのように影響するかを示すために、そして設計支援として、材料の最高の機械的な組み合わせを実現するために実装されました。全体的に、結果は、移植片がレイヤーのプロパティを変更することにより、三層構造を模倣するように設計することができることを示した。

Watch this Scientific Journal Video about Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study at JoVE.com

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

本研究の目的は、動脈壁のネイティブ三層アーキテクチャを模倣することでした。これを達成するために、エレクトロスピニングは3-1（入力 - 出力）ノズルを使用することで採用し、ポリカプロラクトン、エラスチン、コラーゲンのブレンドされた。

ポリカプロラクトン、エラスチン、コラーゲンを使用してネイティブ動脈アーキテクチャを模倣するためにトライレイヤーエレクトロスピニング：予備的研究

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

tri-layered-electrospinning-to-mimic-native-arterial-architecture

Research

JoVE Journal

Bioengineering

12.8K ビュー.  Virginia Commonwealth University. 本研究の目的は、動脈壁のネイティブ三層アーキテクチャを模倣することでした。これを達成するために、エレクトロスピニングは3-1（入力 - 出力）ノズルを使用することで採用し、ポリカプロラクトン、エラスチン、コラーゲンのブレンドされた。

ビデオ: Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

エレクトロスピニングの基礎：最適化ソリューションと装置のパラメータ

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

生物工学

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

ネイティブ、繊維状の長い間隔と分節ロング間隔コラーゲンのin vitro合成

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

皮膚からのI型コラーゲンの調製のための改良された方法

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

変性コラーゲンストランドを画像化する<em&gt;インビボ</em&gt;と<em&gt;生体外</em&gt;リテーナ入りコラーゲン模倣ペプチド写真·トリガブリダイゼーション経由

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

3Dコラーゲンマトリックス中の走化性を調査するために、平面グラデーション拡散システム

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます<em&gt;インビトロ</em&gt;気道壁のモデル

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製<em&gt;インビボ</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

組織工学的応用の制御可能な形態の三次元 Poly(ε-caprolactone) 足場のエレクトロスピニング記述を溶かす

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

心筋細胞のシステム生物学における細胞構造の役割を研究する心筋細胞の構造的に現実的な有限要素の幾何学的なモデルを作成します。

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

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Subtitles

ポリカプロラクトン、エラスチン、コラーゲンを使用してネイティブ動脈アーキテクチャを模倣するためにトライレイヤーエレクトロスピニング：予備的研究