著者らは、 &quot;誘導多能性幹細胞の挙動を制御するための組織工学的足場&quot; NSERCディスカバリーグラント402462を確認したいと思います。

プロトコルを開始する前に注意：フィブリノーゲンは、血液由来のタンパク質であり、したがって、適切な安全訓練が処理する前に完了する必要があります。このプロトコルは2日、所望の幹文化が適切に彼らは播種の準備ができていることを確認するための時間を完了する必要があります。どのくらいのフィブリノゲンは、トリスの3 35ミリメートルペトリ皿を量るための計算条件で400を含む1 24ウェルプレートを生成するのに十分であるTBSの3 mLに溶解フィブリノゲンの110から130 mgを含む（TBS、pH7.4）で緩衝生理食塩水各ウェルに添加フィブリン足場。 1。一日目：フィブリノゲン溶液と一晩透析を作る<ol><li>冷蔵庫の凍結乾燥フィブリノゲンを取り出し、それが室温に来るようにできるように20分間座ってみましょう。 </li><li>トリスの追加3 mLをそれぞれ35ミリメートルシャーレに生理食塩水（TBS）にバッファされます。 12から20までの濃度範囲フィブリノゲン溶液のフィブリノーゲン収率の各プレートには2〜3 mLのmg / mLとし、必要なフィブリノゲン量の合計は、これらの近似に基づいて計算することができます。 </li><li>約100から130 mgのフィブリノーゲン（FG）を秤量し、それぞれの皿にTBSの存在の表面に振りかける。ソリューションに入るために開始するためにフィブリノゲンのために5分間待ってください。蓋付きのペトリ皿をカバーしています。 </li><li> 2時間フィブリノゲンが完全にTBS溶液に移動できるようにするために37℃でペトリ皿をインキュベートします。 </li><li> TBSで濡れた透析チューブ（7000 MWはカットオフ）。チューブとクランプの端倍下端は、透析クランプでシャットダウンします。 </li><li>透析チューブに皿からフィブリノゲン溶液をピペットで。上端の上に折り、透析クランプでシャットダウンクランプ。 </li><li> TBSの4リットル容器にフィブリノゲン溶液を含有する透析チューブを配置します。低速（100回転）に設定し、撹拌プレートを使用してソリューションを混ぜる。 </li><li>少なくとも12時間プレートをかき立てる上で一晩ソリューションを透析。 TBSソリューションは、この期間に変更する必要はありません。 </li></OL&gt; 2。 2日目：足場にフィブリンの足場と幹細胞の播種の重合これらの足場は、幹細胞培養物を接種されるように、このプロセスのために説明されているすべてのステップは滅菌組織培養フード内で実行する必要があります。 <ol><li>適切なサイズのコニカルチューブ（15 mlまたは50 mlのいずれか）に透析チューブと場所からフィブリノゲン溶液を削除します。 </li><li>溶液中での大きな不純物を取り除くために5.0μmのシリンジフィルターとフィルターフィブリノゲン溶液。溶液の体積に応じて、複数のフィルタを使用すると、ソリューション全体を処理する必要があるかもしれません。 </li><li> 50mlコニカルチューブに0.22μmフィルターで滅菌フィルターフィブリノゲン溶液。適切なサイズのピペットを用いて、計算を行うときに、後で使用するためにろ過フィブリノゲン溶液の体積を決定します。溶液の体積に応じて、複数のフィルタの使用は、ENTを処理するために必要があるかもしれません怒り·ソリューションを提供します。 </li><li>分光光度計を用いて280 nmでのフィブリノゲン溶液の吸光度を測定します。 50の希釈係数は1で吸光度を得るためにしばしば必要になります。 </li><li> 1.55は、ヒトフィブリノゲン13 280吸光係数である÷1.55 =（A 280×希釈倍率）[mg / mLでのフィブリノゲン]：次の式を用いてフィブリノゲン溶液中に存在するタンパク質の濃度を計算します。次のTBSの量を計算すると、最初のボリュームに基づいて、フィブリノゲンの11.1 mg / mlに溶液の濃度を希釈する必要がありました。フィブリン足場でのタンパク質の最終濃度は、重合後に10 mg / mLのでしょう。 </li><li>滅菌トロンビンと塩化カルシウム溶液を得る。 24ウェルプレートの最初の行の各ウェルの右側に - ：まず、15μLトロンビン溶液（最終足場の濃度が2 U / mLとなります40 U / mLの濃度）を追加します。次に、50mMのカルシウムCHLの15μLを追加する各ウェルの左側にorideソリューションを提供します。最後に、プレートにフィブリノゲン溶液270μLを追加します。側からのソリューションの混合を確実にするために前に戻って揺れが続く側にプレートを横に振る。 </li><li>混合物は室温で5分間重合を含む行してみましょう。それらが重合として足場はより不透明になります。 </li><li>フィブリン足場、所望の数が重合されたまで、手順2.6および2.7を繰り返します。 </li><li>最後の5分間のインキュベーション後に、足場の完全な重合を可能にするために37℃のインキュベーターの中にプレートを配置します。 </li><li> 1時間のインキュベーションが完了したら、次のステップは、胚様体と種子の足場をすることです。 20μLpipettemenを使用して、フィブリン足場の中心部にある個々の胚様体と場所を選択します。各ウェルは、単一の胚様体が含まれていることを顕微​​鏡で確認してください。 </li><li>と50 mMの塩化カルシウムの5μL：トロンビン溶液5μL（40 U / mLの濃度）を追加各ウェルにフィブリノゲン溶液90μLに続いて、各胚様体の上にソリューションを提供します。解決策は、胚様体をカプセル化し、バブルを形成する必要があります。 </li><li>重合を確実にするためにさらに1時間戻って、​​37℃インキュベーター内で場所のプレート。 </li><li>インキュベーション後、37℃インキュベーターに戻し、各ウェルと場所プレートに適切な細胞培養培地の1 mLを加える。 </li></ol> 3。代表的な結果 図1は、よく胚様体を接種した3Dフィブリン足場培養系を含む個々の側面図の概略を示しています。 図2Aは、マウス胚性線維芽細胞フィーダー層上で培養されているマウスの人工多能性幹細胞の代表的なイメージを示します。これらの細胞は、その後8日レチノイン酸治療プロトコール（ 図2B）previouslとして使用して、胚様体、神経前駆細胞を含む細胞の凝集体を形成するように誘導される図3は、3Dフィブリン足場の内側に培養3日後にiPS細胞由来の胚様体の外観を示しながら、yが14を説明します。同様の結果がマウス胚性幹細胞7を使用して、以前に得られています。さらに、このメソッドは、この培養系の多様性を実証し、レチノイン酸とpurmorphamine 3Dフィブリン足場15の内部を含む別のプロトコルを使用して、文化のiPS由来の胚様体に製造され使用されています。 <img alt="図1。" src="/files/ftp_upload/3641/3641fig1.jpg" /> 図1。よく胚様体で3Dフィブリン足場シードを含む個々の側面図を示す模式図。 <img alt="図2。" src="/files/ftp_upload/3641/3641fig2.jpg" /> 図2。マウス誘導多能性幹（iPS）細胞培養と分化 。 neurにこれらの細胞を区別するらの表現型は、iPS細胞は胚様体（EB）をと呼ばれる細胞の凝集体を生成する懸濁液中で培養される。これらのEBはその後神経分化を誘導するレチノイン酸で処理され、このプロトコルは、以前に胚性幹細胞の神経分化を誘導するために使用されていました。 A）未分化マウスiPS細胞のコロニーは、マウス胚性線維芽細胞フィーダー層上で培養した。 B）懸濁培養でのマウスiPS細胞由来の胚様体は、神経分化を誘導するレチノイン酸による治療後8日目に撮影。スケールバーは100μmである。 <img alt="図3。" src="/files/ftp_upload/3641/3641fig3.jpg" /> マウスiPSの図3の例の結果は、3Dフィブリン足場の内側に培養3日後に体を胚。細胞はフィブリン足場の中に移行し、区別するために始めました。スケールバーは500μmである。

<ol>
	<li>Keung, A. J., Healy, K. E., Kumar, S., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+and+dynamics+of+natural+and+engineered+stem+cell+microenvironments.">Biophysics and dynamics of natural and engineered stem cell microenvironments.</a> Wiley Interdiscip. Rev. Syst. Biol. Med. 2, 49-64 (2010).</li><li>Willerth, S. M., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+stem+cells+and+biomaterial+scaffolds+for+constructing+tissues+and+cell+delivery.">Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery.</a> StemBook. , (2008).</li><li>Lee, J., Cuddihy, M. J., Kotov, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+matrices:+state+of+the+art.">Three-dimensional cell culture matrices: state of the art.</a> Tissue Eng. Part B Rev. 14, 61-86 (2008).</li><li>Ahmed, T. A., Dare, E. V., Hincke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin:+a+versatile+scaffold+for+tissue+engineering+applications.">Fibrin: a versatile scaffold for tissue engineering applications.</a> Tissue Eng. Part B Rev. 14, 199-215 (2008).</li><li>Breen, A., O'Brien, T., Pandit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+as+a+delivery+system+for+therapeutic+drugs+and+biomolecules.">Fibrin as a delivery system for therapeutic drugs and biomolecules.</a> Tissue Eng. Part B Rev. 15, 201-214 (2009).</li><li>Willerth, S. M., Faxel, T. E., Gottlieb, D. I., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+soluble+growth+factors+on+embryonic+stem+cell+differentiation+inside+of+fibrin+scaffolds.">The effects of soluble growth factors on embryonic stem cell differentiation inside of fibrin scaffolds.</a> Stem Cells. 25, 2235-2244 (2007).</li><li>Willerth, S. M., Arendas, K. J., Gottlieb, D. I., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+fibrin+scaffolds+for+differentiation+of+murine+embryonic+stem+cells+into+neural+lineage+cells.">Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells.</a> Biomaterials. 27, 5990-6003 (2006).</li><li>Gorodetsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+Microbeads+Loaded+with+Mesenchymal+Cells+Support+Their+Long-Term+Survival+While+Sealed+at+Room+Temperature.">Fibrin Microbeads Loaded with Mesenchymal Cells Support Their Long-Term Survival While Sealed at Room Temperature.</a> Tissue Eng. Part C Methods. , (2011).</li><li>Barsotti, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+acts+as+biomimetic+niche+inducing+both+differentiation+and+stem+cell+marker+expression+of+early+human+endothelial+progenitor+cells.">Fibrin acts as biomimetic niche inducing both differentiation and stem cell marker expression of early human endothelial progenitor cells.</a> Cell Prolif. 44, 33-48 (2011).</li><li>Park, J. S., Yang, H. N., Woo, D. G., Jeon, S. Y., Park, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenesis+of+human+mesenchymal+stem+cells+in+fibrin+constructs+evaluated+in+vitro+and+in+nude+mouse+and+rabbit+defects+models.">Chondrogenesis of human mesenchymal stem cells in fibrin constructs evaluated in vitro and in nude mouse and rabbit defects models.</a> Biomaterials. 32, 1495-1507 (2011).</li><li>Mooney, R., Tawil, B., Mahoney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+fibrinogen+and+thrombin+concentrations+promote+neuronal+rather+than+glial+growth+when+primary+neural+cells+are+seeded+within+plasma-derived+fibrin+gels.">Specific fibrinogen and thrombin concentrations promote neuronal rather than glial growth when primary neural cells are seeded within plasma-derived fibrin gels.</a> Tissue Eng. Part A. 16, 1607-1619 (2010).</li><li>Liu, H., Collins, S. F., Suggs, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+for+expansion+and+differentiation+of+mouse+embryonic+stem+cells.">Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells.</a> Biomaterials. 27, 6004-6014 (2006).</li><li>Dellenback, R. J., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extinction+coefficient+of+fibrinogen+from+man,+dog,+elephant,+sheep,+and+goat+at+280+mmu.">The extinction coefficient of fibrinogen from man, dog, elephant, sheep, and goat at 280 mmu.</a> Proc. Soc. Exp. Biol. Med. 134, 353-355 (1970).</li><li>Bain, G., Kitchens, D., Yao, M., Huettner, J. E., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+express+neuronal+properties+in+vitro.">Embryonic stem cells express neuronal properties in vitro.</a> Dev. Biol. 168, 342-357 (1995).</li><li>Li, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+ventral+spinal+progenitors+and+motor+neurons+from+human+embryonic+stem+cells+by+small+molecules.">Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules.</a> Stem Cells. 26, 886-893 (2008).</li><li>Lorentz, K. M., Kontos, S., Frey, P., Hubbell, 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=Engineered+aprotinin+for+improved+stability+of+fibrin+biomaterials.">Engineered aprotinin for improved stability of fibrin biomaterials.</a> Biomaterials. 32, 430-438 (2011).</li><li>Willerth, S. M., Rader, A., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+controlled+growth+factor+delivery+on+embryonic+stem+cell+differentiation+inside+fibrin+scaffolds.">The effect of controlled growth factor delivery on embryonic stem cell differentiation inside fibrin scaffolds.</a> Stem Cell Res. 1, 205-218 (2008).</li><li>Chen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+improvement+of+focal+cerebral+ischemia+injury+by+subdural+transplantation+of+induced+pluripotent+stem+cells+with+fibrin+glue.">Functional improvement of focal cerebral ischemia injury by subdural transplantation of induced pluripotent stem cells with fibrin glue.</a> Stem Cells Dev. 19, 1757-1767 (2010).</li></ol>

著者らは、開示することは何もありません。

上記の詳細は、このプロトコルは、マウス胚および誘導多能性幹細胞に特化し、多能性幹細胞培養用の3Dフィブリンの足場を生成するためのメソッドを提供します。この3Dバイオマテリアルベースの培養系は、より正確に生体内で見つかった幹細胞のニッチを模倣し、結果として、それは幹細胞の分化6日にその効果を決定するために生物学的な手がかりをスクリーニングするた...

必要な部品： 化学てんびん pHメータ 組織培養インキュベーター（37℃、5％CO 2） プレートをかき混ぜる 分光光度計 無菌組織培養フードトリス（フィブリノゲンを溶解するための必要性4 Lを加えた十分な）（pH7.4）で緩衝生理食塩水 50mMの塩化カルシウム溶液 滅菌コニカルチューブ（15または50 mL）で 35ミリメートルのペトリ皿 透析チューブ（7,000 MWカットオフ） 透析クリップ 5.0μmのシリンジフィルター 個別包装された無菌の0.22μmのシリンジフィルター 注射器 24ウェル組織培養プレート<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>フィブリノゲン（ヒト） </td><td> Calbioc裾</td><td> 341578 </td><td></td></tr><tr><td>トロンビン（ヒト） </td><td>シグマ</td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

幹細胞は自然に多くの場合、幹細胞のニッチ1と呼ばれる生体内 3次元微小に発生するに記載されています。 3D生体材料足場の内側に培養幹細胞はポリスチレンなどのエンジニアリング交換組織2の方法を使用して、従来の2D培養法上の利点を提供し、正確にこれらの微小環境を模倣する方法を提供します。 2Dの組織培養polystreneは、細胞培養実験の大半に使用されている一方で、3D生体の足場は、より密接な環境で細胞極性のより正確な設置を可能にし、軟組織に似て生化学的および機械的性質を有することにより、生体内で見つかった微小環境を複製することができます。 3の様々な天然由来の合成生体材料足場が幹細胞の成長をサポートするための3D環境として検討されている。合成足場は大きなrを持つように合成することができる一方機械的および化学的性質のアンジュ、しばしば大きく再現性を持っているが、天然の生体材料は、多くの場合、細胞外マトリックスや細胞接着の結合部位が含まれており、容易に細胞培養をサポートして結果として検出されたタンパク質や多糖類で構成されています。血漿から得られた蛋白質フィブリノゲンを重合することによって生成されたフィブリンの足場は、広く、in vitroおよび in vivo 4 の両方の組織工学の様々なアプリケーションのために検討されている。このような足場は、治療因子5を提供するための制御放出システムを組み込むための様々な方法を使用して変更できます。前の仕事は、このような足場が正常に培養胚性幹細胞を使用することができ、この足場ベースの培養系が6,7の内側に播種した幹細胞の分化の様々な成長因子の効果をスクリーニングするために使用することができることが示されています。 このプロトコルの詳細polymerizinのプロセスグラムフィブリンは、トロンビンの酵素活性を用いてフィブリノゲン溶液から足場。プロセスは、重合を阻害するクエン酸塩を除去するフィブリノゲン溶液で一晩透析する工程を含む、完成には2日かかります。これらの詳細な方法は、胚および誘導多能性幹細胞培養のための最適であると判断さフィブリノゲン濃度に依存しています。他のグループはさらに、細胞の種類とアプリケーションの広い範囲のフィブリンの足場を検討した-このアプローチは8から12の汎用性を実証した。

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

この作品の詳細3Dフィブリンの調製は、培養およびplutipotent幹細胞を区別するための足場。このような足場は、同様の薬物送達システムを含むように修正された幹細胞の挙動に及ぼす種々の生物学的化合物の作用をスクリーニングするために使用することができます。

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 ...

preparation-of-3d-fibrin-scaffolds-for-stem-cell-culture-applications

Research

JoVE Journal

Bioengineering

25.1K ビュー.  University of Victoria. この作品の詳細3Dフィブリンの調製は、培養およびplutipotent幹細胞を区別するための足場。このような足場は、同様の薬物送達システムを含むように修正された幹細胞の挙動に及ぼす種々の生物学的化合物の作用をスクリーニングするために使用することができます。

ビデオ: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3次元細胞培養のための自己申告足場

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

生物工学

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

3次元細胞培養モデルの質量分析イメージング用試料調製の戦略

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

関節軟骨細胞培養軟骨の生成のための3Dハイドロゲルの足場

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮細胞の再増殖および腎組織の開発のための齧歯類細胞外マトリックスの足場の調製

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

バイオメディカル応用のための機械的に調整可能な生物活性金属スキャフォールドの作製

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

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

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

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

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

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

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

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

微細と3D微小血管の組立

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...