この作品は、フロリダ国際大学の医工学部門の設備を用いて行った。

1。エレクトロコレクターのセットアップ<ol><li>矩形片にアルミホイルを切った。 </li><li>短冊に矩形片を折るし、テープで平らな金属板（ 図1）に垂直に取り付けます。注：繊維マットの寸法は、ストリップの長さおよび幅に依存する。必要に応じて、このようにして、ストリップの寸法を調整することができる。 </li></ol> 2。高分子溶液の調製<ol><li> PGD​​ポリマーを得100時間、120℃でビーカーに1:1のモル比でグリセロールおよびドデカン二酸（DDA）を混ぜる。 </li><li> 15mlチューブ中の1.5:3:95.5の重量比65％エタノール中でのポリ（エチレンオキシド）（PEO）およびゼラチンを溶解させ、キャップを締めて、撹拌しながら1時間60℃のオーブン中で混合物を加熱それは均一な溶液（基礎溶液）になるまで。 </li><li>エレクトロスピニングの場合は、PGDポリマーと4時06分重量比で、基礎溶液を混合する。注意：PGD濃度は双方向がありグラム繊維径に与える影響。 30％-50％パーセントのPGDはもはや増加しなくなる繊維直径を有する5 cm未満の繊維を製造するのに許容可能である。 </li><li>ポリマー溶液に0.1％のリボフラビンを加え、よく混ぜる。 </li></ol> 3。エレクトロスピニング<ol><li> 18 gの5ミリリットル標準シリンジにポリマー溶液を供給するには、ステンレス製の針を平滑化。 </li><li>シリンジポンプへシリンジを挿入します。 </li><li>金属板への高電圧電源および針に正に荷電リードの接地されたリードを取り付ける。 </li><li> 15センチメートルに針とアルミ箔帯の間の距離を調整します。 </li><li>ストリップの前面に、繊維の凝集を防止するために、水平方向に約15°の角度でシリンジポンプを配置します。 </li><li>シリンジポンプの電源を入れ、0.6ミリリットル/時にポンプの流量を調整します。 </li><li>高電圧電源をオンにして、14.6 kVの動作電圧を設定する。 </li></ol> 4。繊維加工<ol><li>収集が完了すると、架橋のために60分間UV光に繊維マットを露出させる。 </li><li> 100ミリメートルのペトリ皿にアルミ箔ストリップから繊維マットを転送し、殺菌のためさらに20分間UV光にさらす。 </li><li>バイオセーフティキャビネットでは、外科用ブレードを用いて同じ大きさの円形の部分に繊維マットをカットし、24ウェルプレートに作品を配置します。 </li><li>電池プレシーディング処理のために、リン酸緩衝生理食塩水（PBS）1ml中に繊維サンプルを浸漬し、37℃で一晩インキュベートする。 </li><li>翌日、PBSを吸引除去し、慎重に、各ウェルに（材料表内のレシピを参照）を分化培地（DMEM/F12、N2、およびFGF2）の1ミリリットルを加え、3時間37℃でインキュベートする。 </li><li>吸引し分化培地を慎重に、各繊維サンプルに0.2mlのマトリゲルを添加し、30分間37℃でインキュベートする。注：ラミニンはまた、ファイバ被覆のために使用することができる。 20μg/ mlのラミニンの0.2ミリリットルを追加します。ファイバへと3時間室温でインキュベートする。 </li><li>慎重に各ウェルから過剰マトリゲルを削除します。一度分化培地2mlですすいでください。注：繊維試料は、細胞培養の準備ができている。 </li></ol>繊維上5。細胞播種<ol><li>のmES細胞培養皿にして、Accutase 1 mLを加え、37℃で10分間インキュベートする</li><li> 10分後、mES細胞を、培養皿から剥離される。プレートに分化培地4mlを加える。コロニーを破壊するには、上下15ミリリットルチューブとピペット細胞内に浮遊mES細胞を回収（約15倍）。 </li><li>分化培地4ml中の5分間再懸濁細胞を400×gで遠心する。 </li><li>血球計数器を用いて細胞を数える。 15ミリリットルチューブに細胞懸濁液200μlを移すと分化培地で10倍に希釈してください。所定の位置にカバースリップで血球計算板上のチャンバーに希釈した細胞懸濁液15μLを転送します。カウント1ミリメートルの中央広場や顕微鏡下で血球計算板の四隅の正方形のセル。注：ミリリットル当たりの細胞=×10 4希釈倍率X平方あたりの平均回数</li><li>各ウェルに約5×10 4 mES細胞を配置します。ゆっくりと繊維マットを水抜きからソリューションを防ぐために、代わりにほかの側にオフにスライドする、繊維試料の途中に細胞懸濁液をドロップします。 </li><li>各ウェルに分化培地1 mLを加え、37℃のインキュベーターにプレートを保持し、5％CO 2細胞の付着、増殖および分化を可能にする。 </li><li>各ウェルから古い培地を吸引し、一日おきに新鮮な分化培地1mlと交換してください。 </li></ol> 6。細胞生存率<ol><li>各ウェルから古い培地を吸引除去する。 </li><li>培養培地でレサズリン蛍光試薬の1/10の容積を混合し、各ウェルに1ミリリットルを加える。 </li><li> Incub37℃、直接光から保護し、4時間、5％CO 2で食べ。 </li><li> 4時間後、96ウェルプレートに各ウェルから試薬100μlの3倍を転送し、次にサンプルは560EX nm/590EMフィルタ設定を用いた蛍光分光光度計で測定される準備ができている。 </li></ol> 7。リアルタイムPCR <ol><li>培養14日後に、サンプルに溶解緩衝液を追加し、繊維試料上の細胞からトータルRNAを単離する。 </li><li>逆転写により20μlの反応スケールでcDNAを合成した全RNAの4μlを使用する。 </li><li> 10μlのマスターミックス（2×）、0.2μlの染料、8μlのヌクレアーゼフリー水、1μlのcDNA、および1μlのプライマー（本研究で用いたプライマーを表1に示します）：各鏡筒内のPCR反応液を調製する。 </li><li> PCRプログラムをセットアップします。 95℃で2時20分、1サイクルB。 95℃で3秒間→60℃で1分、40サイクルC。 95℃15秒、1サイクルd。 60℃で1分、1サイクルE。 95℃15秒、1サイクル。相対的な遺伝子発現レベルを決定するために、比較C T法を選択する。 </li><li> PCRが終了した後、StepOneソフトウェアでのリアルタイムPCRの結果を分析し、Excelに結果をエクスポート。 </li></ol>

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J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tough+biodegradable+elastomer.">A tough biodegradable elastomer.</a> Nature biotechnology. 20, 602-606 (2002).</li><li>Panunzi, S., De Gaetano, A., Mingrone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approximate+linear+confidence+and+curvature+of+a+kinetic+model+of+dodecanedioic+acid+in+humans.">Approximate linear confidence and curvature of a kinetic model of dodecanedioic acid in humans.</a> American Journal of Physiology-Endocrinology And Metabolism. 289, (2005).</li><li>Park, 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=Apparatus+for+preparing+electrospun+nanofibers:+designing+an+electrospinning+process+for+nanofiber+fabrication.">Apparatus for preparing electrospun nanofibers: designing an electrospinning process for nanofiber fabrication.</a> Polymer Internationa l. 56, 1361-1366 (2007).</li><li>Barnes, C. 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利害の対立が宣言されていません。

単純コレクタまたは現在いくつかの用途のための所望の長さおよび繊維マットのサイズを取得する制限を増加させるエレクトロスピニングするために使用されるコレクタを回転させる複雑さの限界。さらに、培養皿または他の基板に接地コレクタから繊維を転送することは挑戦5である。本報告では、接地された集電体にアルミニウム箔ストリップを取り付けることにより簡単に行わ?...

エレクトロスピニングは、マイクロ·ツー·ナノサイズの繊維足場を作製するための効果的な処理方法の一つである。エレクトロスピニングの基本原理は、針の先端と接地されたコレクタとの間に高電圧を印加して、針の開口部に保持される溶液のテーラーコーンを含む。溶液中の静電反発力が表面張力を克服したときに、荷電流体ジェットは、針先端から排出され、溶媒の蒸発と空気を通って移動し、最終的に接地された集電体上に堆積される。シリンジポンプは、紡糸口金から出てくる溶液の連続流を提供し、電界紡糸繊維のこのように複数のコピーが短時間で製造することができる。コレクタに到達するために紡糸口金を出るの過程の間に、帯電したジェットは、ポリマー溶液の粘度及び表面張力を含むパラメータの数、electrostatiに記載の伸縮及び泡立て受けるC溶液の力、及び外部電場との相互作用など2。 電界紡糸プロセスにおいては、集電体は、マイクロ·ツー·ナノファイバーが堆積することができる導電性基板として機能する。本研究では、繊維集新しいタイプの所望のサイズ（長さ×幅）で繊維マットを得るように設計した。伝統的に、アルミニウム箔を集電体として使用されるが、他の基板に平坦な表面から繊維を転送することは困難である。伝統的なコレクタから無傷の繊維マットを収穫することの難しさは、電界紡糸繊維がコレクターの表面に強固に付着するということが主な原因だった。そこで、短冊にアルミニウム箔片を折り畳み、金属平板に垂直に取り付けることによってコレクタを修飾。電界紡糸繊維は、ストリップの先端と簡単に別substratに転送することができる金属板との間の領域にわたって延伸されるE。 熱架橋エラストマーポリマーへの関心が急速にため、ポリ（グリセロールセバケート）（PGS）、2002年3において、加硫ゴムに似ています。PGSと同様にポリエステルを導入しましたロバート·ランガーのグループの先駆的な仕事の成長、私たちが開発に成功しているされているグリセロールおよびドデカン二酸の熱縮合によりポリ（グリセロール-ドデカン）（PGD）とその独特な形状記憶特性1を示した。より硬い合成材料であるポリ（ヒドロキシブチレート）又はポリ（L-ラクチド）（それぞれが250MPaおよび660メガパスカルのヤング率）とは異なり、PGDは、温度が37℃以上である1.08メガパスカルのヤング率を有するゴムのようなエラストマー性を示すその場末梢神経（0.45メガパスカル）に近い試合で、C、。加えて、PGDは、生分解性であり、分解時間は、グリセロールおよびドデカン二酸の比率を変えることによって微調整することができる。ドデカン二酸は12炭素のサブです2端子カルボキシル基、HOOC（CH 2）10 COOHとスタンス。セバシン酸及びドデカン二酸等が偶数番号のジカルボン酸は、アセチル-CoAおよびトリカルボン酸（TCA）/（クエン酸）サイクルを入力するように代謝され得る。ジカルボン酸の代謝産物、スクシニル-CoAは、糖新生前駆体およびTCA回路4の中間体である。したがって、いくつかの研究は、それらが、特に病的状態では、経腸および非経口栄養のための代替燃料の基質として利用できることが示唆された。そのガラス転移温度が31°Cであるため、また、PGD、従ってそれは室温及び体温で異なる機械的性質を示す、ユニークな形状記憶を呈する。要するに、PGDは、神経組織に類似した機械的特性を持つユニークな弾性特性を示す、生分解性、生体適合性である;従って、それは神経組織工学用途に適した材料である。このプロトコルでは、エレクトロ大沈着領域にまたがる長い繊維が、PGDから新設計のコレクタを経由して作製した。ファイバー足場は、マウス多能性幹細胞の増殖および分化を支持することができる。

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Glycerol</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7757</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dodecanedioic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Gelatin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Poly (ehtylene oxide) (PEO)</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">182028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Riboflavin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">132350250</td>
			<td style="width:167px;">0.10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Mouse embryonic stem cells</td>
			<td style="width:175px;">GlobalStem</td>
			<td style="width:119px;">GSC-5002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Matrigel</td>
			<td style="width:175px;">Becton Dickinson</td>
			<td style="width:119px;">356234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">DMEM/F12</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:119px;">SH30272.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">N2 supplement&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">17502048</td>
			<td style="width:167px;">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">FGF2</td>
			<td style="width:175px;">Stemgent</td>
			<td style="width:119px;">03-0002</td>
			<td style="width:167px;">10ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Accutase</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A11105-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">10010-031&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Resazurin fluorescence dye&#160;</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">62758-13-8&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">SV Total RNA Isolation System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">Z3100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoScript Reverse Transcription System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A5000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoTaq&#129; qPCR Master Mix</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A6001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Syringe pump&#160;</td>
			<td style="width:175px;">Fisher scientific</td>
			<td style="width:119px;">14-831-200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:268px;">High voltage power source&#160;</td>
			<td style="width:175px;">Spellman High Voltage Electronics Corporation</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">SL30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">UV light</td>
			<td style="width:175px;">Philips</td>
			<td style="width:119px;">308643</td>
			<td style="width:167px;">15W/G15T8</td>
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			<td height="21" style="height:21px;width:268px;">Synergy HT Multi-Mode Microplate Reader</td>
			<td style="width:175px;">BioTek</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Perkin Elmer GeneAmp PCR System 9600</td>
			<td style="width:175px;">Perkin Elmer</td>
			<td style="width:119px;">8488</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:268px;">StepOne Real-time PCR System</td>
			<td style="width:175px;">Applied Biosystems</td>
			<td style="width:119px;">4376357</td>
			<td style="width:167px;"></td>
		</tr>
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</table>

electrospun fibrous scaffolds of poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells

組織工学用途のために、生分解性および生体適合性の足場の調製は、最も望ましいが困難なタスクである。様々な製造方法の中で、エレクトロスピニングは、そのシンプルさと汎用性に最も魅力の一つです。さらに、電界紡糸ナノ繊維は、細胞の生存および増殖のための追加の支持を確保する天然の細胞外マトリックスのサイズを模倣する。本研究では、エレクトロスピニングのために新たに設計されたコレクタを使用してポリ（グリセロール-ドデカン）（PGD）1という名前の新 ​​たな生分解性と生体適合性ポリマーのためのより大きな沈着領域にまたがる長繊維の製造の実現可能性を示した。 PGD​​は、従って、それが神経組織工学用途に適している、神経組織と同様の機械的特性を有する独特の弾性特性を呈する。繊維性足場材料を製造するための合成および製作のセットアップは、簡単で再現性の高い、安価であった。生体適合性に試験、マウスの胚性幹細胞由来の細胞はに付着し、エレクトロスピニングPGD繊維上で成長できた。要するに、このプロトコルは、マウス胚性幹細胞由来の神経系細胞の増殖を支持するためにPGDの電界紡糸繊維を製造するための多用途の製造方法を提供した。

エレクトロスピニングの主要なコンポーネントが図1に示されている。大型繊維マットは、典型的には、垂直に取り付けられたアルミニウム箔ストリップと金属平板を介して得た。 図2は、集電体の設計および電気紡糸繊維マットを示す。幅及び長さは、異なる用途のために調整することができる。 PGD​​ポリマーおよび基底溶液混合物で作られた繊維の長さは10cmま?...

Watch this Scientific Journal Video about Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells at JoVE.com

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

ポリ（グリセロール - ドデカン）という小説の生分解性ポリマーから新たに設計されたコレクタを経由して、より大きな沈着領域にまたがるエレクトロ長繊維（PGD）の合成及び製造が報告された。繊維は、マウス多能性幹細胞に由来する細胞の増殖を支持することができた。

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 ...

electrospun-fibrous-scaffolds-polyglycerol-dodecanedioate-for

Research

JoVE Journal

Bioengineering

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

10.8K ビュー.  Florida International University. ポリ（グリセロール - ドデカン）という小説の生分解性ポリマーから新たに設計されたコレクタを経由して、より大きな沈着領域にまたがるエレクトロ長繊維（PGD）の合成及び製造が報告された。繊維は、マウス多能性幹細胞に由来する細胞の増殖を支持することができた。 

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

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

動脈組織工学におけるエラストマーのPGSの足場

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

生物工学

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

幹細胞培養アプリケーションのための3Dフィブリン足場の調製

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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 ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

開発、展開、および<em生体内で&gt;</em&gt;ヒト胚性幹細胞（ヒトES細胞）からヒトNK細胞のモニタリングとや人工多能性幹細胞（性IPSC）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

繊維状足場に微小球を離すエレクトロ成長因子

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

ポリ（εカプロラクトン）エレクトロ糸にヒト間葉系幹細胞の添付ファイルの最適化

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

繊維組織における階調の電界紡糸ナノファイバー足場

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

脱細胞肺足場を使用してESC由来のマウス気道上皮細胞の生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

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 ...