資金は、国立衛生研究所（EB 001013）、カナダ自然科学工学研究評議会と保健研究のカナダの協会によって提供されていました。私たちは、彼女の専門知識のために博士はAPマクギガンに感謝し、モジュールの開発に役立つ。

1）チューブの調製<ol><li>ポリエチレンチューブの2〜3メートルの長さをカット（0.76ミリメートル内径x 1.22ミリメートルOD）。 </li><li>チューブの一端に20 G1の針を通します。 </li><li>コンバータセルフシールパウチ（7 1 / 2&quot;× 13&quot;）のコイルチューブと場所。チューブの両端は、袋の未舗装の端に置かれ、ガス滅菌のテープでテーピングしてください。 </li><li>シールの袋とガスは滅菌する。 </li></ol>コラーゲンの2）中和注：コラーゲンの1mlは、ポリエチレン管の約2 mを埋めます。 <ol><li> 15 mlコニカルチューブにチューブの所望の長さに必要なコラーゲンの必要量を配置し、氷上に置きます。 </li><li>繰り返しソリューションをピペッティングすることによりチューブとミックスに10倍のα- MEMあたりのmLのコラーゲンの128のULを追加します。注：（1）ソリューションは、酸性化されたコラーゲンにα- MEMのような黄色が混合されている電源を必要と（2）混合時に気泡を最小限にしようとする。 </li><li>コラーゲンは7.4のpHになるまで0.8Mの重炭酸ナトリウムを添加することによりコラーゲンを中和する。黄色のコラーゲン溶液が淡いピンクの色が消えるまで、pHの一般的な見積もりの​​ために重炭酸ナトリウムを加える。注：これは一般的にコラーゲンの各MLあたり30〜60μLを取る。 </li><li>中和コラーゲンとして使用する準備ができるまでゲル4で保たれていない場合になります氷上で中和コラーゲンを維持℃に</li></ol>細胞の3）埋め込み（オプション） 注：細胞を溶解あたり20万個の細胞へのmL当たり100万の間の細胞の濃度で埋め込むことができる細胞の種類にもよります。あなたが埋め込むするセルに必要な培地を使用してください。 <ol><li>細胞から培地を取り除き、5mLのPBSで洗浄する。 </li><li> PBSを削除し、3 mLのトリプシン- EDTAを加える。 </li><li> 37℃で5分間のためにトリプシン- EDTAで細胞をインキュベート</li><li> 7 mLのメディアで細胞を再懸濁します。 </li><li>細胞を数える。 </li><li> 15 mLコニカルチューブに埋め込むための細胞の必要数を転送する。 </li><li> 5分間、300 xgでペレットは、細胞。 </li><li>細胞ペレットから培地を吸引除去する。 </li><li>中和コラーゲンに細胞を再懸濁し、氷上に置きます。 </li></ol> 4）コラーゲンのゲル化は、 <ol><li>生物学的安全キャビネット内でポリエチレンチューブの入った袋の密封端をカット。 </li><li>滅菌ピンセットを用いてチューブの一端に20G針に3 mlの注射器を取り付けます。袋にチューブを保持します。 </li><li>先端が滅菌ピンセットを用いてコニカルチューブの一番下になるまで中和コラーゲンにチューブのもう一方の端を差し込みます。コニカルチューブを氷上で保管してください。 </li><li>シリンジのプランジャーは、徐々に後退によるポリエチレンのチューブにコラーゲンを引き出します。 注：コラーゲンはすぐにその後にチューブに引き込まれている場合、気泡がコラーゲンで形成される。 </li><li>チューブを介してコラーゲンを引っ張っした後、チューブの外20G針を引っ張ると袋に両端を戻す。テープバッグがシャットダウンします。 </li><li> ℃で60分間で37袋をインキュベートする。コラーゲンは細胞が含まれている場合は、毎15分かけて袋を裏返し。その細胞は、モジュールの片側にセトリングしていないこと。 </li></ol> 5）チューブの切断はゲル状になったコラーゲンを含む<ol><li>切削装置とチューブを保持するトレイ用オートクレーブヘッダとブレード。 </li><li>切削装置を組み立てます。 </li><li>場所は、デバイスを切断の前に滅菌トレイのチューブと切断装置にチューブをロードする。 </li><li>カットモジュールを収集する切削装置の出口でメディアの25 mLを含む50mLのコニカルチューブを設定します。 注：モジュールは、メディアのFBSのないチューブから分離しないようにコラーゲンのみのモジュールに対してもFBSを含む使用メディア。モジュールが含まれている場合は細胞が埋め込まれ、細胞に必要なメディアを使用してください。ある場合はモジュール内に組み込まれた細胞は、内皮細胞用培地を使用します。 </li><li>チューブの2mmの長さをカットしてチューブを切断する切断装置を設定します。 </li><li> 37モジュールをカット° 50 mLコニカルチューブに60分間Cをインキュベートする。 </li></ol> 6）チューブからコラーゲンモジュールの取り外し<ol><li> 10秒のためのチューブ内のモジュールを含む渦50 mLコニカルチューブ。 注：破損を避けるために高い細胞密度で埋め込まれたモジュールを分離するときに優しく。 </li><li>モジュールは、チューブの底に沈降してみましょう。約5分かかります。 </li><li>広口10mlの血清学的ピペットを使用すると、15 mLコニカルチューブに決済モジュールを転送する。 </li><li>ステップ6.1から6.3倍を繰り返します。 </li><li>非組織培養処理されたプレートに内皮細胞または転送モジュールとコートのモジュールに、手順7に進みます。 </li></ol> 7）コーティング内皮細胞とモジュール<ol><li>番目のメディアを5 mL、15 mLコニカルチューブの底にモジュールをセトルeは、細胞を埋め込まれた。 </li><li>内皮細胞から培地を取り除き、5mLのPBSで洗浄する。 </li><li> PBSを削除し、3 mLのトリプシン- EDTAを加える。 </li><li> 37℃で5分間のためにトリプシン- EDTAで細胞をインキュベート</li><li> 7 mLのメディアで細胞を再懸濁します。 </li><li>細胞を数える。コニカルチューブの底にパックされたモジュールのmL当たり5 × 10 6内皮細胞を必要とする。 </li><li>ペレットを5 mLの内皮細胞培地中で5分間再懸濁しますため、300 xgで細胞。 </li><li>モジュールに内皮細胞を含む培地を5mLを追加。 注：これは、我々は両方の細胞型の生存と機能のために働くことが判明している2つのメディアタイプの50:50比率を与える。使用にそれを使用する前に、二つの細胞タイプの表現型を適切に管理するように定式化共培養の培地をテストします。 </li><li>静的および動的に内皮細胞でモジュールをインキュベートする。 </li><li>静的インキュベーションミックス反転による内皮細胞とのモジュールと37℃で直立管をセット℃で15分のため。繰り返します。 </li><li>軽く37℃チューブを揺らすことによって動的にシード内皮細胞℃で60分間。 </li><li>二回静的インキュベーションを繰り返します。 </li><li>非組織培養は、治療をプレートにモジュールを配置し、37℃で一晩インキュベート℃、 </li><li>次の日の代わりに新たな非組織培養のモジュールがアタッチされていない内皮細胞を除去するために新鮮な培地（共培養系を用いた場合50:50）にプレートを処理した。 </li><li>彼らがモジュールの表面の100％をカバーするまで、内皮細胞で被覆されているモジュールをインキュベートする。これは通常、約7日かかります。 </li></ol> 8）代表的な結果： 彼らは最初のチューブから削除されたときのモジュールは、円筒形になります。モジュールが組み込まれた細胞および/を含むまたは内皮細胞で被覆されている場合、それらは体積で50％まで収縮し、楕円形（図1）を開発することができます。モジュールはまた、光学顕微鏡（図1）で見たときより高密度とより不透明になります。また、内皮細胞は、モジュールの表面上にコンフルエントのときにVE -カドヘリンの免疫蛍光染色（図2）で見られるタイトジャンクショ​​ンの形成がある。物質移動の解析は、モジュールが不十分な酸素の輸送に起因する壊死性コアを開発することなく、高い細胞密度（8 × 10 7細胞/ cm 3の ） をサポートできることを明らかに大きな組織の中にしばしば問題とされている（大口径の例：モジュール、D = 1.4しましたMM）（図3）。 <img src="/files/ftp_upload/2177/2177fig1.jpg" alt="図1" /> 図1：モジュールの製造と収縮。 HUVEC - C（内皮細胞株）シードを後3日中に発生したモジュールの収縮はかなり小さく、モジュールの直径と長さ（P &lt;0.001）（）の結果。埋め込まれたHepG2細胞は一様に、製造時にモジュール内で分配と高い生存率を（B）保持された。 [細胞は緑のライブ、死んだ細胞赤] [コーストフィン2010年から適応] <img src="/files/ftp_upload/2177/2177fig2.jpg" alt="図2" /> 図2：モジュールの表面にコーティングされた10日RAEC後のECタイト細胞間結合のVE -カドヘリン免疫蛍光染色。スケールバー= 50μmの。 <img src="/files/ftp_upload/2177/2177fig3.jpg" alt="図3" /> 図3：典型的なモジュール（0.76ミリメートル初期直径）と大型モジュールのマッソントリクローム染色（1.4ミリメートル初期直径）は、酸素の拡散の制限の効果を実証。製造後7日間は、死細胞の多数は生存細胞の唯一の薄いリムを（〜200μmの厚さ）を残して、大規模なモジュール（右下のパネル）のコア内に形成していた。逆に、小規模なモジュールは、生きている細胞の均一かつ高いディストリビューションを保持。 [モジュールは、HepG2細胞を包埋し、内皮細胞で被覆された。] [コーストフィン2010年から撮影] <img src="/files/ftp_upload/2177/2177fig4.jpg" alt="図4" /> 図4：poloxamine上（A）HMEC - 1シード-コラーゲンのモジュール。 poloxamine -コラーゲンモジュールを播種の1日後に円筒形の形状を保持し、コラーゲンのみモジュールに比べて限定された細胞の添付ファイルのプロパティがあります。スケールバー= 200μmの。 PLGAベースの生分解性ミクロスフェアを含むコラーゲンのモジュールの（B）光顕微鏡像。スケールバー= 500μmの。 <img src="/files/ftp_upload/2177/2177fig5.jpg" alt="図5" /> 図5：in vitro試験の例。 （A）血管新生アッセイ：血管内皮細胞を接種したモジュール上の毛細血管のような岩は、簡単に脂肪由来幹細胞とのインキュベーションの5日後に検出および定量することができる。 （B）のモジュール上でライブ/デッドアッセイの共焦点顕微鏡画像生きている細胞のために泰寧は緑であり、死んだ細胞のために赤いです。 <img src="/files/ftp_upload/2177/2177fig6.jpg" alt="図6" /> 図6：埋め込 ​​まれた間葉系幹細胞と内皮細胞の表面層を含むコラーゲンモジュール。モジュールは、マイクロ流体室で7日間せん断応力（〜0.64 DYN / cm 2）のために暴露し、接触点での融合を示し始めるれた。内皮細胞は、vWFを（茶色）で染色し、間葉系幹細胞核は青色（H＆E）が表示されている。スケールバー=100μmの。 <img src="/files/ftp_upload/2177/2177fig7.jpg" alt="図7" /> 図7：脂肪由来幹細胞（ASC）およびヒト微小血管内皮細胞で被覆されたを含むコラーゲンのモジュールの何百もの（HMEC）脂肪再生アプリケーションのためのin vivoでの ASC / HMEC相互作用を研究するためにマウスの皮膚の下に注入される。

<ol>
	<li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules.">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li><li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells.">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li><li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats.">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li><li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells.">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li><li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+[corrected].">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion.">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+tissue+engineering:+fabrication+of+a+gelatin-based+construct.">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering.">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+criteria+for+a+modular+tissue-engineered+construct.">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules.">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li><li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen/poloxamine+hydrogels:+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells.">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li></ol>

利害の衝突は宣言されません。

我々は、異なる種類の細胞1月11日に埋め込 ​​まれたモジュールを使用していくつかの異なるミクロ組織を作製した。我々は成功し、主心筋細胞、膵島、脂肪幹細胞と間葉系幹細胞と同様にしたHepG2、NIH 3T3およびクローン9肝臓の細胞を含むいくつかの細胞株が組み込まれています。我々は、ラット大動脈内皮細胞、ヒト臍帯静脈内皮細胞とヒト微小血管内皮細胞を含む内皮細胞のい...

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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>Intramedic tubing PE60</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>427416</td>
<td>Different diameter of tubing can be used to change the diameter of the modules</td>
</tr>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>Purcol acidificed collagen, 3 mg/mL</td>
<td>&nbsp;</td>
<td>Cedarlane</td>
<td>5005-B</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>20G needle</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>305175</td>
<td>Diameter of the needle needs to be similar to the diameter of the tubing</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>15 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>Convertors Self-Seal Pouch 7 1/2&#x201D; x 13&#x201D;</td>
<td>&nbsp;</td>
<td>Cardinal Health</td>
<td>92713</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Wide Tip serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357504</td>
<td>&nbsp;</td>
<tr>
<td>10 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>5 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357543</td>
<td>&nbsp;</td>		</tbody>
		</table>
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fabrication of micro-tissues using modules of collagen gel containing cells

このプロトコルは、モジュールと呼ばれるミクロ組織の型の製造を説明します。モジュールのアプローチは、均一な、スケーラブルで血管が新生組織を生成します。モジュールは、コラーゲンだけでなく、他のゲル化または架橋可能な材料から成っていることができる。彼らは、製造時に直径で長さが約2mmと0.7 mmであるが、埋め込まれたセルまたは時のモジュールが内皮細胞で被覆すると大きさに縮小。個別のモジュールが組み込まれた細胞は酸素と他の栄養素の拡散の制限範囲内であるが、モジュールがperfusableな大きな組織を形成するために一緒にパックすることができるくらい小さいです。彼らは複雑な組織を形成するために一緒にパックされる前に別の種類の細胞が内に埋め込ままたはモジュール上にコーティングすることができますので、これらの組織は、建設中のモジュラーです。モジュールを作るために3つの主要なステップがあります：（1）コラーゲンを中和し、その中に細胞を包埋、管の（2）ゲル化コラーゲンをし、内皮細胞とのモジュールと（3）コーティングモジュールを切断。

Watch this Scientific Journal Video about Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells at JoVE.com

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

埋め込まれた細胞とその表面を含むモジュールと呼ばれる円筒状のコラーゲンゲルを、使用して、ミクロ組織の作成は、内皮細胞で被覆されている。

コラーゲンゲル含有する細胞のモジュールを使用してマイクロ組織の作製

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

fabrication-micro-tissues-using-modules-collagen-gel-containing

Research

JoVE Journal

Bioengineering

12.6K ビュー.  University of Toronto. 埋め込まれた細胞とその表面を含むモジュールと呼ばれる円筒状のコラーゲンゲルを、使用して、ミクロ組織の作成は、内皮細胞で被覆されている。

ビデオ: Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

動的なマスク投影リソグラフィーを用いたニューラル培養システムのためのマイクロパターンハイドロゲルの作製

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

生物工学

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 ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

三次元インクジェット印刷技術を用いて、ヒト軟骨組織の作製

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

圧縮されたコラーゲンで細分化した組織：シングル段再建修復のための細胞含有Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

ゴールドナノロッドを含む細胞車の作製と光音響分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

ジェル支援補水を使用した巨大なサイズのポリマーソームを形成します

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

マクロを含む光の組織ファントムの作製と評価

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

準備、精製、および脂肪酸含有リポソームを用いる

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

ヒト間葉系幹細胞によって Falcarindiol を含む新しい脂質被覆ナノ粒子の取り込み

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

リゾ脂質含有温度感受性リポソームのマイクロ流体生産

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...