この作品は、JLIとヒース助成R01AR056624の国立研究所にアメリカ心臓協会博士号を取得する前のフェローシップによってサポートされていました。

A.単一のポリマーエレクトロスピニング<ol><li>エレクトロスピニング溶液を調製する前に、数日間37℃で溶解して脱イオン水でイルガキュア2959（I2959）、、光開始剤の0.5重量％溶液を作る。光反応性ポリマーが使用されていない場合、この手順は必要ありません。 </li><li> 2重量％MeHA、3重量％の最終濃度で溶液を調製メタクリル化ヒアルロン酸（MeHA、バーディックらを参照してください。合成のため）、ポリ（エチレンオキシド）（PEO、900 kDaの）、および脱イオン水でI2959を組み合わせるPEO、および0.05重量％I2959。それが透明になるまで溶液を混合するために渦を使用してください。ポリマーの種類および濃度、ならびに使用される溶媒は、所望の足場の特性に応じてこのステップで変更することができます。 </li><li>シリンジに液を移し、最後に18ゲージ、長さは6インチ、平滑末端の針を取り付けます。 </li><li> 1.2 mLの/ hrの速度で排出するプログラムシリンジポンプ。デバイスへの注射器と針を挿入します。 </li><li>収集装置に高電圧電源の接地リード線を取り付けます。ランダムに分散した繊維は平らな金属板を使用して収集し、または整列繊維を収集するために〜10 m / sで回転するマンドレルを使用する。針に正に帯電したリードを取り付けます。エレクトロスピニング装置の概略図1を参照してください。 </li><li> 、両者の間を15cmの距離があるような針や収集装置を調整します。 </li><li>シリンジポンプの流量を開始します。流体が針の先端に視覚化されるときは、電源をオンにし、22 kVに電圧を設定します。 </li><li>コレクションは、（薄膜の数分から、最大厚いマットのための24時間まで）の完了後、収集装置から足場を削除し、溶剤の完全除去を確実にするために一晩真空下で保管してください。 </li><li>走査型電子顕微鏡（SEM）を用いて繊維の形態を視覚化する、代表的な足場が整列し、非整列の両方の構造については図1に示されています。 </li></ol>注：サンプル流量、収集装置までの距離、および電圧は、ポリマーと溶媒の組み合わせに依存しており、通常はSEMで足場の形態を観察することによって、各システム用に最適化されている必要があります。 B.の光架橋と光パターニング<ol><li>足場のマットから5mm × 5mmの試料を切り出した。 </li><li>きれいなスライドガラスで覆うとバインダークリップで両端をクリッピング、足場上で直接フォトマスクを配置し、スライドガラスをカバーフォイルで各足場を置くことによって、架橋のために準備する。注：4つのサンプルは一度に行うことができる、とパターンが必要ない場合は、パターンのない透明性、架橋のために使用することができます。 </li><li>パージ足場は、窒素のチャンバー内に設定します。架橋を阻害することができる酸素、より構造がフリー状態にしておくことが重要です。 </li><li> 5分間コリメートアダプター付き〜10 MW / cm 2の 365nmの光の下で窒素チャンバ内の場所に足場のセットアップ。 図2の回路図を参照してください。 </li><li>各足場を取り外し、12ウェルプレートの場所。 </li><li> 37水と場所の蒸発を防ぐために、各ウェルに精製水2 mLをパラフィルムプレート追加℃で24時間。水を3回変更してください。 </li><li>光学顕微鏡下で孔形成を可視化する（ 図2の例を参照）。足場は、使用する準備は完了です。 </li></ol>注：光架橋は、ポリマーの多くの種類のために必要ではない、しかし、光パターニングは、光反応性ポリマーを使用することができます。 蛍光ファイバーの可視化とC.デュアルポリマーエレクトロスピニング<ol><li> 90％エタノールでPEO（200 kDa）の5重量％の溶液を調製します。 50℃より前のエレクトロスピニングに少なくとも2時間、700 rpmで攪拌​​。 </li><li>シリンジにPEOのソリューションを移し、エレクトロスピニング溶液中に10 mg / mLの濃度の最終濃度のためにDAPIを追加。光から保護するために、アルミホイルでシリンジを包みます。 </li><li>また、エレクトロスピニング溶液中で25μMの最終濃度methacryloxyethylチオカルバモイルローダミンB（MeRho、683.24グラム/モル）を追加する場合を除き、ステップA2で説明した同じ溶液を調製します。光から保護するために、アルミホイルでシリンジを包みます。 </li><li>注意深くマンドレルの表面にメタクリルガラス製カバースリップを（Khademhosseini らを参照してください。）確保するためにスコッチテープを使用してください。注：繊維は、メタクリルガラス製カバースリップに縛らになります。 </li><li>シリンジのいずれかにルアーロックの添付ファイル付きのシリコンチューブの5 mm長部分の一端を添付して、針にもう一方の端を取り付けます。シリンジポンプにシリンジを挿入し、送風機の穴から針を刺します。マンドレルの反対側のサイト上で他の注射器とあおぐ人で繰り返します。 fannersはlを翻訳マンドレルのengthと結果足場の両方のポリマーの平等な分配を確保するために使用されています。 図3の回路図を参照してください。 </li><li>適切に表1にしたがってセクションで説明されているパラメータを調整します。針の先端がマンドレルを中心にされていることを確認します。 表1。デュアルポリマーエレクトロパラメータ <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 注射器 </td><td> ニードルチップ（cm）にあおぐ人 </td><td> マンドレル（cm）に針の先端 </td><td> 流量（mLの/時） </td><td> 印加電圧（kV）の </td></tr><tr><td> MeHA </td><td> 6 </td><td> 15 </td><td> 1.2 </td><td> 22 </td></tr><tr><td> PEO </td><td> 6 </td><td> 10 </td><td> 1.2 </td><td> 15 </td></tr></tbody></table> </li><li>すべてが適切に配置されると、ライトをオフにしてマンドレルとシリンジポンプをオンにします。シリンジからアルミホイルを取り外します。流体は、両方の針の先端上に表示されているときに、同時にfannersでの電源とプラグをオンにします。 </li><li>収集が完了すると、電源装置とマンドレルをオフにしてfannersを抜いてください。慎重にカミソリの刃を使用してカバースリップとテープを取り外します。 </li><li>ローダミンおよびDAPI用フィルターを装備した蛍光顕微鏡を用いて繊維を可視化する。 図3の例を参照してください。注：蛍光繊維は、短期間（&lt;3分）のためのエレクトロスピニングは、しかしながら、このプロセスは、特にガラス製カバースリップを使用せずに、厚い構造を作成するために時間を無制限に拡張することができるときに表示するには、最も明確である。 </li></ol>足場上でD.シーディングセル<ol><li>適切なサイズのウェルプレートの個々のウェルに接続されているエレクトロポリマーを使用して各カバースリップを配置。注：繊維と細胞間相互作用は、メタクリルガラス製カバースリップの上にエレクトロスピニングによって簡単に表示されている。また、MeRhoは再び繊維の可視化に使用することができます。 </li><li>溶剤の完全除去と副産物すべての可能な浸出性を確保するために一晩PBSで足場をインキュベートする。 </li><li>井戸からPBSを削除します。 </li><li>足場を殺菌するために、30分間層流フードで殺菌灯の下に配置します。完全な足場はシードされている場合、別の30分間殺菌灯の下で上の足場と場所を反転させる。 </li><li>標準細胞培養を行い、（例えば、6,000細胞cm -2）の所望の細胞密度で濃縮された細胞懸濁液を準備する。例えば、22㎜× 22 mmのカバースリップのために細胞懸濁液100 mLを使用してください。 1時間培養器に置きます。 </li><li>各ウェルに細胞培養培地の適切な量を追加。インキュベーターに戻して足場を置きます。 </li><li>市販のライブ/デッドキットを用いて細胞を染色。注：このようなDAPI​​（細胞核）と蛍光標識ファロイジン（アクチンストレスファイバー）などの細胞の染色の他のタイプは、細胞を可視化に適用することができます。 </li><li> TRITCおよびFITC用のフィルターを装備した蛍光顕微鏡を用いて細胞と繊維を可視化する。 図4の例を参照してください。 </li></ol>代表的な結果： <img src="/files/ftp_upload/1589/1589figure1tmb.jpg" border="0" alt="図1" /> 図1非整列足場の形成（上）とアライン足場の形成（下）のデバイスのセットアップを示す回路図。足場の各タイプの例走査型電子顕微鏡の画像が表示されます。スケールバー=5μmの。してください<a href="https://www.jove.com/files/ftp_upload/1589/1589figure1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 <img src="/files/ftp_upload/1589/1589figure2tmb.jpg" border="0" alt="図2" /> 図2。パターニング方法の概略図。パターンは、光架橋時の光源と足場の間にフォトマスクを配置し、離れて未反応のポリマーを洗浄することによって形成される。孔形成および凍結乾燥後の足場のフォトマスクとSEM像。スケールバー= 100μmの。してください<a href="https://www.jove.com/files/ftp_upload/1589/1589figure2.jpg">ここをクリックして</a>図2の拡大バージョンを参照すること。 <img src="/files/ftp_upload/1589/1589figure3tmb.jpg" alt="図3" border="0" /> 図3マルチポリマーエレクトロの設定と必要な処理パラメータの回路図、ならびに2つのファイバの集団（例：赤MeHAと青PEO）の混合物の代表的な蛍光画像のマルチポリマー足場を形成するために同時にエレクトロスピニングされた。スケールバー= 100μmの。してください<a href="https://www.jove.com/files/ftp_upload/1589/1589figure3.jpg">ここをクリックして</a>大きい版？を参照する図3のn。 <img src="/files/ftp_upload/1589/1589figure4tmb.jpg" border="0" alt="図4" /> 図4ヒト間葉系幹細胞とエレクトロの繊維との相互作用の例デッド/ライブ染色像。スケールバー= 100μmの。してください<a href="https://www.jove.com/files/ftp_upload/1589/1589figure4.jpg">ここをクリックして</a>図4の拡大バージョンを参照すること。

<ol>
	<li>Burdick, J. A., Chung, C., Jia, X., Randolf, M. A., 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=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>Baker, B. M., Gee, A. O., Metter, R. B., Nathan, A. S., Marklein, R. A., Burdick, J. A., Mauck, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29, 2348-2358 (2008).</li><li>Ifkovits, J. L., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: Photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue Engineering. 13, 2369-2385 (2007).</li><li>Khademhosseini, A., Eng, G., Yeh, J., Fukuda, J., Blumling, J., Langer, R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment.">Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.</a> J. Biomed Mater Res A. 79A, 522-532 (2006).</li><li>Mauck, R. L., Baker, B. M., Nerurkar, N. L., Burdick, J. A., Li, W. J., Tuan, R. S., Elliott, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+on+the+Straight+and+Narrow:+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration.">Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration.</a> Tissue Engineering B. 15, 171-193 (2009).</li><li>Sill, T. J., Von Recum, H. a. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , (2009).</li></ol>

エレクトロスピニングは、高分子から繊維質足場を準備するために使用されていました。ヒアルロン酸に基づいて光架橋の足場は、露光が架橋のために必要とされる説明の例として使用した。このようなMeHAような反応性マクロマー、を使用すると、以前に強化された細胞分布を実証しているチャンネルは、マクロとミクロ多孔足場を形成する光架橋時のマスクを用いて足場に編入された。ま...

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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>DAPI</td>
<td> Reagent</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>&nbsp;</td>
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEO 200 kDa</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>17503</td>
<td>&nbsp;</td>
<tr>
<td>PEO 900 kDa</td>
<td>Reagent</td>
<td>Sigma</td>
<td>189456</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxethyl thiocarbamoyl rhodamine B</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591-100</td>
<td>Prepare stock solution in DMSO</td>
</tr>
<tr>
<td>Live/Dead Stain Kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Contains Calcein (stains live cells green) and ethidium homodime (stains red dead cells)</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>Equipment</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Power Source</td>
<td>Equipment</td>
<td>Gamma High Voltage</td>
<td>ES30P-5W</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Motor</td>
<td>Equipment</td>
<td>Triem Electric Motors, Inc</td>
<td>0132022-15</td>
<td>Must attach to a custom built mandrel</td>
</tr>
<tr>
<td>Tachometer</td>
<td>Equipment</td>
<td>Network Tool Warehouse</td>
<td>ESI-330</td>
<td>Use to monitor mandrel speed</td>
</tr>
<tr>
<td>Omnicure UV Spot Cure System with collimating adapter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>Silicone Tubing</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51135K151</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Female Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K293</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Male Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K143</td>
<td>&nbsp;</td>
<tr>
<td>Needles</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14-825-16H</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Equipment</td>
<td>Corning</td>
<td>2875-22</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrospinning fibrous polymer scaffolds for tissue engineering and cell culture

組織工学の分野が発展するにつれて、より複雑な臓器や組織の要件（例えば、力学と血管）に対処するために、より適切な素材と加工技術を生成するために非常に大きな需要があります。エレクトロスピニングは、ネイティブの細胞外マトリックスのアーキテクチャと大きさのスケールを模倣繊維状足場を作成するための一般的な手法です。繊維が細胞挙動を指示するために使用することができるので、これらの繊維状の足場は、幹細胞の分化（Mauckによる大規模なレビューをすべて見る含め、また、細胞培養の基質として有用である<em&gt;ら。</em&gt;とシル<em&gt;ら。</em&gt;詳細については）。この記事では、ポリマーのエレクトロスピニングの一般的なプロセスを説明し、例として、electrospin光照射による架橋が可能な反応性ヒアルロン酸（参照Ifkovits<em&gt;ら。</em&gt;光架橋材料の見直しのため）。我々はまた、光パターニング及びマルチポリマー足場の形成など、さらなる処理能力をご紹介。光パターニングは、細胞浸潤と組織分布を向上させるチャンネルとマルチスケール空隙率と骨格を作成するために使用することができます。マルチポリマー足場は良い細胞​​浸潤のために気孔を合わせた含む足場の性質（力学と劣化）を、調整するのに便利です。さらに、これらの技術は成功した組織工学的構造の開発に必要な手がかりを提供する複雑な足場を作成するためのポリマーと反応性マクロマーの広い配列を含むように拡張することができます。

Watch this Scientific Journal Video about Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture at JoVE.com

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

組織工学と細胞培養のためのポリマーをエレクトロスピニングのプロセスは、この記事で取り上げられています。特に、光パターニング及びマルチポリマーエレクトロスピニングの追加の処理能力を持つ光反応性マクロマーのエレクトロスピニングが記載されている。

組織工学と細胞培養のための繊維状重合体の足場をエレクトロ

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

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JoVE Journal

Biology

21.4K ビュー.  University of Pennsylvania. 組織工学と細胞培養のためのポリマーをエレクトロスピニングのプロセスは、この記事で取り上げられています。特に、光パターニング及びマルチポリマーエレクトロスピニングの追加の処理能力を持つ光反応性マクロマーのエレクトロスピニングが記載されている。

ビデオ: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Experimental Approaches to Tissue Engineering

組織工学への実験的アプローチ

生物学

Micro-scale Engineering for Cell Biology

細胞生物学のためのマイクロスケールのエンジニアリング

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

三次元細胞培養用チップサイズの足場の微細加工

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

インタビュー：幹細胞研究のためのバイオリアクターと浮上し、修正3D -足場

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

組織工学のための3Dハイドロゲルの細胞のカプセル化

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

エンジニアリング細胞透過性のタンパク質

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

イモリのレンズ再生を研究するために培養虹彩色素上皮細胞のためのシステム

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

強化されたバイオセーフティのためのポリマー複合エコトロピックレンチウイルスによるヒト細胞のトランスダクション

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

細胞培養で誘導し、低酸素のテスト

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...