Name: easy manipulation of architectures in protein-based hydrogels for cell culture applications
Uploaded: 2017-08-04T11:00:00.000+00:00
Duration: 8 min 50 s
Description: Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

著者は、「バイオインスパイアード材料合成」フレームワーク (バイオマット-14) の金融支援財団はバーデン = ヴュルテンベルク州を感謝したいです。

1. ゲルの調製
<ol>
	<li>BSA の 200 mg をミックス 1 ml の脱イオン H2O 20% (w/v) BSA ストック (ストック溶液) を作成します。</li>
	<li>ミックス 165 μ THPC ソリューション (134 mg/mL) 4.835 ml THPC を作成する純水の貯蔵液 (原液 B)。</li>
	<li>KCSSGKSRGDS (1,111.1 g/mol) ペプチド (または同等の細胞接着ペプチド) の 1 mg の重量を量るし、滅菌 H2O 10 mg/mL 解 (原液 C) を得るための 100 μ L で希釈しています。 注: この手順はオプションであり、のみ細胞培養用ヒドロゲルを意味する場合が含まれる必要があります。</li>
	<li>96 ウェル プレートの下部を取り外して取り外し可能なプラスチック製のラップ。 注: ここで使用される板、下部簡単に削除できます; 各ウェルの底に圧力を適用することによって薄いプラスチック製の底が単に抜け落ちます。</li>
	<li>BSA 原液のミックス 100 μ L (A) と THPC の 100 μ L 在庫ソリューション (B) (オプション: 機能、細胞接着のヒドロゲルの原液 C の 4 μ L) ハイドロゲルの 200 μ L を取得する 96 ウェル プレートで。重合後制服ハイドロゲルを保証するため、少なくとも 5 回を上下にピペッティングによるコンポーネントをミックスします。</li>
	<li>すべてのゲルは、正しく重合させるまで約 10 分の室温 (RT) で 96 ウェル プレートを配置します。</li>
	<li>慎重に、ゆっくりと、プレートの底からプラスチック製のラップを削除します。</li>
	<li>小さなスタンプを使って 96 ウェル プレートからゲルを押し、滅菌 PBS、pH 7.4 で 1.5 mL チューブにそれらを転送します。 注: ヒドロゲル直径約 4 mm と 8 mm の高さの円筒形があります。</li>
	<li>リン酸緩衝生理食塩水 (PBS) 数ヶ月まで 4 ° c に、ゲルを格納します。</li>
</ol>
2. 凍結、ゲル
<ol>
	<li>無菌脱イオン H2O の 500 μ L で 1.5 mL チューブを記入し、キャップを取り外します。ヘラを使って 1.5 mL の反応管に、ゲルを転送します。</li>
	<li>各バイアル用パラフィン フィルムで少なくとも 3 層でしっかりと 1.5 mL チューブをラップします。針を使用すると、チューブからのガス放出を可能にするのにフィルムに小さな穴を開けます。</li>
	<li>次の手順のいずれかを行います。
	<ol>
		<li>水とゲルが完全に凍結を保証する 5 分間液体窒素ソリューションにバイアルを転送します。液体窒素から除去した後すぐに材料が融解するを防ぐために乾燥機の凍結バイアルを転送します。 注: この手順の結果毛穴について半径 10-15 μ m。</li>
		<li>ゆっくりと、ゲルを凍結するために一晩-20 ° C でバイアルをしてください。-20 ° C から除去した後すぐに材料が融解するを防ぐために乾燥機の凍結バイアルを転送します。 注: この手順の結果毛穴について半径 50-60 μ m。</li>
	</ol>
	</li>
	<li>24 h (およびヒドロゲルへの水の完全な蒸発) 後凍結乾燥機からそれを削除することによって、材料を解凍します。 注: 細孔径は、共焦点レーザ走査型顕微鏡 (手順 5、「ハイドロゲル可視化」) を分析できます。</li>
</ol>
3. 粒子の溶出
<ol>
	<li>1.1 1.5 の手順で説明するように、ハイドロゲルを準備します。</li>
	<li>直接コンポーネントを混合した後飽和 (36 mg/mL) が発生するまで塩化ナトリウムを追加します。白色沈殿物としてソリューションに塩の結晶を見ることができるまでは、塩を追加します。</li>
	<li>96 ウェル プレートをシェーカーに転送、重合が行われる (約 10 分) までを振る。1.7 1.8 の手順で説明するよう、プレートから、ハイドロゲルを削除します。</li>
	<li>ハイドロゲル テンプレートからすべての塩を溶出するシェーカー (20 rpm) で滅菌水で RT で、少なくとも 24 時間のヒドロゲルを孵化させなさい。数ヶ月まで PBS のための 4 ° C でゲルを格納します。</li>
</ol>
4 チャネル形成
<ol>
	<li>手順 1 で説明したゲルを準備します。挟み撃ちを使用したソリューションからハイドロゲルを削除、高吸水紙で余分な水分を除去、ドライアイスのブロックの上に置きます。</li>
	<li>30 のゲルを凍結 s、ブロックから注意深く取り外します。ハイドロゲル; に損傷を与えない慎重にブロックを掻きにヘラを使用します。ヒドロゲルを 1.5 mL チューブに転送し、37 ° C で一晩乾かしてください</li>
</ol>
5. ハイドロゲルの可視化
<ol>
	<li>ローダミン B 原液を準備するには、10 mL の PBS のローダミン B の 1 mg を希釈します。0.001 mg/mL の濃度に達するまで PBS のローダミン原液を希釈してシリアル希釈を準備 (希釈倍率: 100)</li>
	<li>ストレージ ソリューション (例えば、ステップ 2.4 から。) からヒドロゲルを削除し、0.01 mg/mL ローダミン B 溶液 1 mL と 1.5 mL チューブにそれを転送します。染色したローダミン B 溶液で一晩ハイドロゲル</li>
	<li>次の日は、10 mL の PBS (pH 7.4) の少なくとも 3 時間洗浄を可視化することは、ハイドロゲルを転送します。</li>
	<li>よく μ スライド 8 にハイドロゲルを転送し、PBS でそれをカバーします。</li>
	<li>刃を使用して (約 0.5 mm の高さ) ハイドロゲルの小さなスライスをカットします。</li>
	<li>514 の波長でハイドロゲルを可視化する顕微鏡、共焦点レーザーを使用して nm (目的: EC 計画 Neofluar 40 x/1.30 油 DIC M27、平面スキャン モード: 514 nm、およびズーム: 1 X)。</li>
</ol>
6. 細胞文化の可能性
<ol>
	<li>無菌フィルター ソリューション (A、B、および C) を 0.45 μ m のフィルターで在庫すべて。</li>
	<li>1.1-1.4 ゲル重合前に細胞接着ペプチドを含む手順の説明に従って、ハイドロゲルを準備します。</li>
	<li>コンポーネントを混合した後すぐにピペットの混合物 μ スライド 8 に下部は完全に覆われているまで。 注: この手順する必要があります実行する迅速かつ直接コンポーネントを混合した後数分のスライドで重合が行われるよう。</li>
	<li>細胞接着と標準的な細胞培養の技術を用いた単一細胞懸濁液を入手への道。あらかじめ加温、滅菌 DMEM 培ウシ胎児血清 (FBS、10% (w/v))、ペニシリン ・ ストレプトマイシン (1% (w/v))、非必須アミノ酸ソリューション (MEM、1% (w/v)) と補完にセルを転送します。</li>
	<li>ノイバウアーの商工会議所および慎重にピペット 200 μ L のハイドロゲル表面にセル (2 x 105セル/cm2) の目的の数を数えるとセルをカウントします。</li>
	<li>蓋付きも μ スライド 8 をカバーし、インキュベーター (37 ° C、5% CO2) に譲渡すること。37 ° C で少なくとも 4 時間インキュベートします。</li>
	<li>携帯電話の添付ファイルの少なくとも 4 時間後 200 μ L 滅菌培養 PBS で 2 回細胞を洗います。</li>
	<li>RT で 10 分の 3.7% (v/v) ホルムアルデヒドの 200 μ L でセルを修正し、PBS で 2 回洗う (〜 200 μ L)。 注: は、ホルムアルデヒドを処理するときに適切な個人用保護具を着用します。</li>
	<li>0.1% トリトン X を PBS で 2 回 5 分洗浄のための 200 μ L でセルを permeabilize します。</li>
	<li>195 μ L の PBS とメタノール株式の 5 μ L を混合してした汚れで暗闇の中 20 分のセルをそれを追加セルにファロイジン ローダミンで細胞を染色します。PBS で 2 回洗浄します。</li>
	<li>514 で共焦点顕微鏡を用いた細胞接着特性を調べる nm (目的: EC 計画 Neofluar 40 x/1.30 油 DIC M27、平面スキャン モード: 514 nm、およびズーム: 1 X)。適切なソフトウェアを使用して画像を分析 (材料の表を参照してください)。</li>
</ol>
7. ヒドロゲルの物性
<ol>
	<li>膨張率です。
	<ol>
		<li>完全に少なくとも 1 日の 37 ° C でゲルを乾燥します。</li>
		<li>各ハイドロゲルの重さし、正確な重量に注意してください。</li>
		<li>1.5 mL の PBS で 2 mL の反応管を埋めます。</li>
		<li>この 2 mL の反応管に、ハイドロゲルを転送し、完全に PBS で浸します。</li>
		<li>PBS PBS と平衡に到達する RT で、少なくとも 2 日間、ハイドロゲルを残します。</li>
		<li>3 日後、ハイドロゲルをソリューションから削除し、ハイドロゲル表面から余分な水分を削除する紙ティッシュで拭くこと。</li>
		<li>ヒドロゲルの重量を量る。</li>
		<li>次の数式を使用して膨張比を計算します。 
		<img alt="Equation" src="/files/ftp_upload/55813/55813eq1.jpg"> Wdは乾燥ゲル (ステップ 7.1.2。) の重量と Wsは湿潤ゲル (ステップ 7.1.7) の重量です。膨潤率パーセントを得るため 100 を乗算します。</li>
	</ol>
	</li>
	<li>pH と温度安定性。
	<ol>
		<li>15 mL の反応管に 5 mL の PBS を転送します。</li>
		<li>水酸化ナトリウムと塩酸持参適切な温度 (例えばRT、37 ° C、または 80 ° C) への解決策 (例えばpH 2、7、または 10) 適切な pH の解決策を調整します。</li>
		<li>適切なソリューションにその安定性を調査するのには、ハイドロゲルを転送します。必要な場合は、pH を調整してください。 注: すべてヒドロゲル必要がありますが完全に腫れてこの時点 (膨張比を参照) 材料の膨潤による重量の誤った解釈を防ぐために。</li>
		<li>特定の時間間隔の後、ハイドロゲルを (例えば、 2 日前までの各 1 時間) ソリューションから削除、余分な水分を除去し、それの重量を量る非常に吸収性紙ティッシュで乾燥します。</li>
	</ol>
	</li>
	<li>酵素分解。
	<ol>
		<li>300 U トリプシンとペプシン、製造元の指示に従って在庫酵素溶液を準備します。</li>
		<li>(例えば1.8 のステップから) ハイドロゲルを適切なソリューションに転送します。 注: すべてのゲル必要があります完全に腫大しているこの時点で重量の誤った解釈を防ぐために。</li>
		<li>特定の時間間隔の後、ハイドロゲルを (例えば、時間ごと) のソリューションから削除、余分な水分を除去し、それの重量を量る非常に吸収性紙ティッシュで拭くこと。</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Geckil, H., Xu, F., Zhang, X., Moon, S., Demirki, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hydrogels+as+extracellular+matrix+mimics.">Engineering hydrogels as extracellular matrix mimics.</a> Nanomedicine. 5 (3), 469-484 (2011).</li><li>Liu, Y., Chan-Park, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomimetic+hydrogel+based+on+methacrylated+dextran-graft-lysine+and+gelatin+for+3D+smooth+muscle+cell+culture.">A biomimetic hydrogel based on methacrylated dextran-graft-lysine and gelatin for 3D smooth muscle cell culture.</a> Biomaterials. 31 (6), 1158-1170 (2010).</li><li>Raic, A., R&#246;dling, L., Kalbacher, H., Lee-Thedieck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+macroporous+PEG+hydrogels+as+3D+scaffolds+for+the+multiplication+of+human+hematopoietic+stem+and+progenitor+cells.">Biomimetic macroporous PEG hydrogels as 3D scaffolds for the multiplication of human hematopoietic stem and progenitor cells.</a> Biomaterials. 35 (3), 929-940 (2014).</li><li>Wong Po Foo, C. T. S., Lee, J. S., Mulyasasmita, W., Parisi-Amon, A., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-component+protein-engineered+physical+hydrogels+for+cell+encapsulation.">Two-component protein-engineered physical hydrogels for cell encapsulation.</a> Proc Nat Acad Sci USA. 106 (52), 22067-22072 (2009).</li><li>Zhu, J., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+properties+of+hydrogel+tissue-engineering+scaffolds.">Design properties of hydrogel tissue-engineering scaffolds.</a> Expert Rev Med Devic. 8 (5), 607-626 (2011).</li><li>Samaryk, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+Approach+to+Develop+Porous+Hydrogels+with+a+Regular+Pore+Distribution+and+Investigation+of+their+Physicomechanical+Properties.">Versatile Approach to Develop Porous Hydrogels with a Regular Pore Distribution and Investigation of their Physicomechanical Properties.</a> J Appl Polym Sci. 114 (4), 2204-2212 (2009).</li><li>Li, X. J., Valadez, A. V., Zuo, P., Nie, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+3D+cell+culture:+potential+application+for+tissue-based+bioassays.">Microfluidic 3D cell culture: potential application for tissue-based bioassays.</a> Bioanalysis. 4 (12), 1509-1525 (2012).</li><li>Park, H., Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Swelling+Ratio+of+Injectable+Hydrogel+Composites+on+Chondrogenic+Differentiation+of+Encapsulated+Rabbit+Marrow+Mesenchymal+Stem+Cells+In+Vitro.">Effect of Swelling Ratio of Injectable Hydrogel Composites on Chondrogenic Differentiation of Encapsulated Rabbit Marrow Mesenchymal Stem Cells In Vitro.</a> Biomacromolecules. 10 (3), 541-546 (2010).</li><li>Stokols, S., Tuszynski, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fabrication+and+characterization+of+linearly+oriented+nerve+guidance+scaffolds+for+spinal+cord+injury.">The fabrication and characterization of linearly oriented nerve guidance scaffolds for spinal cord injury.</a> Biomaterials. 25 (27), 5839-5846 (2004).</li><li>Sung, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Impact+of+2D+and+3D+Fibroblast+Cultures+on+In+Vitro+Breast+Cancer+Models.">Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models.</a> PLoS One. 8 (10), 1-13 (2013).</li><li>Tsai, E. C., Dalton, P. D., Shoichet, M. S., Tator, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+hydrogel+guidance+channels+facilitate+regeneration+of+adult+rat+brainstem+motor+axons+after+complete+spinal+cord+transection.">Synthetic hydrogel guidance channels facilitate regeneration of adult rat brainstem motor axons after complete spinal cord transection.</a> J Neurotrauma. 21 (6), 789-804 (2004).</li><li>Shoichet, M. S., Li, R. H., White, M. L., Winn, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+hydrogels+used+in+cell+encapsulation:+An+in+vitro+comparison+of+alginate+and+agarose.">Stability of hydrogels used in cell encapsulation: An in vitro comparison of alginate and agarose.</a> Biotechnol Bioeng. 50 (4), 374-381 (1996).</li><li>Chiu, Y. -. C., Kocag&#246;z, S., Larson, J. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+physical+and+mechanical+properties+of+porous+poly+(ethylene+glycol)-co-(L-lactic+acid)+hydrogels+during+degradation.">Evaluation of physical and mechanical properties of porous poly (ethylene glycol)-co-(L-lactic acid) hydrogels during degradation.</a> PloS One. 8 (4), e60728 (2013).</li><li>Jonker, A. M., L&#246;wik, D. W. P. M., van Hest, J. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-+and+Protein-Based+Hydrogels.">Peptide- and Protein-Based Hydrogels.</a> Chem Mater. 24 (5), 759-766 (2012).</li><li>Bodenberger, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+bread+and+beer:+whole+cell+protein+extracts+from+baker's+yeast+as+a+bulk+source+for+3D+cell+culture+matrices.">Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.</a> Appl Microbiol Biot. 101 (5), 1-11 (2016).</li><li>Bodenberger, N., Paul, P., Kubiczek, D., Walther, P., Gottschalk, K. E., Rosenau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+cheap+and+easy+to+handle+protein+hydrogel+for+3D+cell+culture+applications+a+high+stability+matrix+with+tunable+elasticity+and+cell+adhesion+properties.">A novel cheap and easy to handle protein hydrogel for 3D cell culture applications a high stability matrix with tunable elasticity and cell adhesion properties.</a> Chem Sel. 1 (7), 1353-1360 (2016).</li><li>Agarwal, S., Wendorff, J. H., Greiner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrospinning+technique+for+biomedical+applications.">Use of electrospinning technique for biomedical applications.</a> Polymer. 49 (26), 5603-5621 (2008).</li><li>Hong, J., deMello, A. J., Jayasinghe, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-electrospraying+and+droplet-based+microfluidics:+control+of+cell+numbers+within+living+residues.">Bio-electrospraying and droplet-based microfluidics: control of cell numbers within living residues.</a> Biome Mater. 5 (2), 21001 (2010).</li><li>Jayasinghe, S. N., Irvine, S., McEwan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+electrospinning+highly+concentrated+cellular+suspensions+containing+primary+living+organisms+into+cell-bearing+threads+and+scaffolds.">Cell electrospinning highly concentrated cellular suspensions containing primary living organisms into cell-bearing threads and scaffolds.</a> Nanomedicine. 2 (4), 555-567 (2007).</li><li>Selimovi&#263;, &. #. 3. 5. 2. ;., Oh, J., Bae, H., Dokmeci, M., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+strategies+for+generating+cell-encapsulating+hydrogels.">Microscale strategies for generating cell-encapsulating hydrogels.</a> Polymers. 4 (3), 1554-1579 (2012).</li><li>Annabi, N., Nichol, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+porosity+and+microarchitecture+of+hydrogels+for+tissue+engineering.">Controlling the porosity and microarchitecture of hydrogels for tissue engineering.</a> Tissue Eng Part B Rev. 16 (4), 371-383 (2010).</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 (1), 61-86 (2008).</li><li>Bodenberger, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methods+for+pore+generation+and+their+influence+on+physio-chemical+properties+of+a+protein+based+hydroge.">Evaluation of methods for pore generation and their influence on physio-chemical properties of a protein based hydroge.</a> Biotech Rep. 12, 6-12 (2016).</li><li>Raja, S. T. K., Thiruselvi, T., Mandal, A. B., Gnanamani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+and+redox+sensitive+albumin+hydrogel:+A+self-derived+biomaterial.">pH and redox sensitive albumin hydrogel: A self-derived biomaterial.</a> Sci Rep. 5, 15977 (2015).</li><li>Hennink, W. E., van Nostrum, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+crosslinking+methods+to+design+hydrogels.">Novel crosslinking methods to design hydrogels.</a> Adv Drug Deliver Rev. 64, 223-236 (2012).</li><li>Chung, C., Lampe, K. J., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrakis(hydroxymethyl)+phosphonium+chloride+as+a+covalent+cross-linking+agent+for+cell+encapsulation+within+protein-based+hydrogels.">Tetrakis(hydroxymethyl) phosphonium chloride as a covalent cross-linking agent for cell encapsulation within protein-based hydrogels.</a> Biomacromolecules. 13 (12), 3912-3916 (2008).</li><li>Cal&#243;, E., Khutoryanskiy, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+hydrogels:+A+review+of+patents+and+commercial+products.">Biomedical applications of hydrogels: A review of patents and commercial products.</a> Eur Polym J. 65, 252-267 (2015).</li><li>Huang, H., Herrera, A. I., Luo, Z., Prakash, O., Sun, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+transformation+and+physical+properties+of+a+hydrogel-forming+peptide+studied+by+NMR,+transmission+electron+microscopy,+and+dynamic+rheometer.">Structural transformation and physical properties of a hydrogel-forming peptide studied by NMR, transmission electron microscopy, and dynamic rheometer.</a> Biophys J. 103 (5), 979-988 (2012).</li><li>Draghi, L., Resta, S., Pirozzolo, M. G., Tanzi, 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=Microspheres+leaching+for+scaffold+porosity+control.">Microspheres leaching for scaffold porosity control.</a> J Mater Sci. 16 (12), 1093-1097 (2005).</li><li>Whang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Bone+Regeneration+with+Bioabsorbable+Scaffolds+with+Novel+Microarchitecture.">Engineering Bone Regeneration with Bioabsorbable Scaffolds with Novel Microarchitecture.</a> Tissue Eng. 5 (1), 35-51 (1999).</li><li>Ziv, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tunable+silk-alginate+hydrogel+scaffold+for+stem+cell+culture+and+transplantation.">A tunable silk-alginate hydrogel scaffold for stem cell culture and transplantation.</a> Biomaterials. 35 (12), 3736-3743 (2014).</li><li>Butruk-Raszeja, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Athrombogenic+hydrogel+coatings+for+medical+devices--Examination+of+biological+properties.">Athrombogenic hydrogel coatings for medical devices--Examination of biological properties.</a> Colloid Surface B. 130, 192-198 (2015).</li><li>L&#252;, S., Li, B., Ni, B., Sun, Z., Liu, M., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+injectable+hydrogel+for+three-dimensional+cell+culture:+chondroitin+sulfate+bioconjugated+with+poly(N-isopropylacrylamide)+synthesized+by+RAFT+polymerization.">Thermoresponsive injectable hydrogel for three-dimensional cell culture: chondroitin sulfate bioconjugated with poly(N-isopropylacrylamide) synthesized by RAFT polymerization.</a> Soft Matter. 7 (22), 10763 (2011).</li><li>Ruoslahti, E., Pierschbacher, 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=New+perspectives+in+cell+adhesion:+RGD+and+integrins.">New perspectives in cell adhesion: RGD and integrins.</a> Science. 238 (4826), 491-497 (1987).</li></ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

The production of macroporous matrices can be beneficial to many different fields. It has high technical and economic potential due to the defined structure of the hydrogel and the ability to control and tune specific material properties. However, the introduction of supramolecular structural elements, such as pores or channels, to a 3D template might influence the overall properties of a material, such as the swelling ratio or the stiffness. This can result in the undesired decomposition, degradation, or breakdown of th...

ゲルは、大量の水をバインドできる不溶性の 3 D ネットワークを形成する材料です。そのような材料は、生きている細胞の優れた環境を提供できます。現在、三次元ハイドロゲル構造の生成とその化学的・物理的特性を調整するプロセスの開発、関心が、高まっています。これが達成されれば、細胞の増殖および細胞挙動の操作用のテンプレートは生成された1,2,3,4をすることができます。これらの 3 D 構造だけでなく環境を作成するより自然でリアルな二次元従来が、彼らよりも明らかに幹細胞の成長のための新たな可能性や腫瘍モデル5。異なる材料は、主に6ゲルの気孔のサイズに依存する特性の範囲を所有しています。毛穴は、電池文化、組織工学、幹細胞指向性の成長に重要な役割を再生します。たとえば、マトリックスを通して拡散酸素と栄養と十分な量は、セル7に到達できる必要があります。その一方で、有害な代謝産物は、できるだけ早く削除する必要がある、細胞の成長のための十分な領域が利用可能な7をする必要があります。その結果、材料、およびこうして細孔径のプロパティ深刻な行列の潜在的な利益と可能なアプリケーションに影響します。材料の特性に応じて、異なるセル成長過程は、神経構造の形成を含む、3 D の細胞の文化において発生することが成長と皮膚や骨の細胞の分化のような肝細胞や線維芽細胞2,3,8,9,10,11の特別な細胞ラインの監督の成長。物質の可能性のあるアプリケーションに影響を与えるもう一つの重要なポイントは、外部刺激12に向かってその安定性です。たとえば、ヒドロゲルへの細胞培養媒体または人間の体の機械的完全性を維持しなければなりません。
近年、激化、3 D 細胞培養ハイドロゲルに関する研究し、システム13の 3 D アーキテクチャを解決する多くの研究を行った。高い安定性を発揮し、簡単に合成および化学的に変更できるため化学的に合成された成分から成るハイドロゲルを調べた最も一般的5(朱ら2011 のレビューを参照してください)。しかし、タンパク質は多くの有益な特性をある: としていわゆる「精密高分子」彼らは生体適合性;ある定義された長さ;彼らも比較的容易に修正。ターゲット サイト14,15の大規模な数であります。この点では、特異性の高い、革新的な構造は、多くの分野でのアプリケーションの生成できます。本研究では蛋白質ベースのゲル16は、材料の 3 D アーキテクチャに影響を与える確立した方法の能力を発揮する使用されました。さらに、性と細孔生成への適用も検討しました。
簡単な方法と材料科学のさまざまな分野から、専門性の高い高度な技術の両方を含む、3 D 構造を変更するさまざまな方法があります。広範な技術は、明確に定義された構造17を生成するエレクトロスピニングの使用です。請求繊維電界印加によるソリューションから取得され、酸素への露出に固めます。このように、数ミクロンまで数ナノメートルの範囲の繊維を作り出すことができます。サイズ、構造、およびマトリックス内の気孔の分布を調整するための追加のテクニックは、ソフト ・ リソグラフィー、フォトリソグラフィ、流体を中心、エレクトロ スプレー、およびバイオ印刷18,19,20。これらの技術の重大な欠点は、特定の高価な装置や特別な化学薬品材料への依存関係です。さらに、これらの技術と経験が頻繁に蛋白質ベースの材料に直接譲渡と化学物質とメソッドの多くは、互換性のある細胞ではないです。
その一方で、多くの技術は特別な装置は、簡単かつ安価な適用し、再現するそれらを作るには頼らないでください。構造操作のため普及している方法は、溶液キャスト法21,22,23です。粒子は重合反応前に加えてゆくソリューションを飽和に配布されます。毛穴が材料内に残る、重合後、希釈や pH 変化などの条件の変化は粒子の溶媒和に します。塩、砂糖、パラフィン、ゼラチン、チョークなど、これらの技術で使用される化学物質は、安価で容易に入手できます。凍結、膨潤ゲルに固定されます。真空下で液相の後続の昇華は、23,24,25を実行します。ネットワークから水昇華は材料の特定の立体構造を維持するために十分な穏やかです。発泡ガス、重合が行われる、ゲル21孔を残している間に、ガスとソリューションはストリーミングされます。サイズと孔の分布は、ガスの流れによって調整できます。
タンパク質ゲルを形成、BSA はテトラキス (ヒドロキシメチル) ホスホニウム塩塩化 (THPC) 第一級アミンとリンカーの 4 武装分子26の水酸基との間に共有結合の形成を許可する Mannich 型反応と反応しました。可能な有害な中間体は、反応が発生した後、材料の余分な洗浄によって削除されます。
本研究では、BSA ベースの素材を操作し、細孔のサイズを調整するさまざまなテクニックを治療の可能性を紹介します。各技法使用できますあらゆる研究室で、世界中、特別な機器は必要ありません。さらに、パラメーターの異なるような膨潤率、酵素分解性、pH 安定性、温度感受性を検討され、それぞれに他と比較して特に尊重さまざまなテクニックの影響 3 D アーキテクチャの世代に。最後に、材料が修飾と細胞接着ペプチドの細胞培養の材料の適用の可能性を調査します。2 つの異なるモデル細胞ラインを使用した: A549 と MCF7。

<table><tbody><tr><td>Phosphate Buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (high glucose)</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>11140-050</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>10270-106</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>MEM Nonessential Amino Acid Solution</td><td>Sigma Aldrich</td><td>M7145-100ML</td><td></td></tr><tr><td>Trypsin EDTA 0.05% Phenol Red</td><td>Thermo Fisher Scientific</td><td>25300062</td><td></td></tr><tr><td>Ethanol 99.8%, verg&amp;auml;llt</td><td>&amp;Ouml;lfabrik Schmidt</td><td>2133</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Carl Roth&amp;nbsp;</td><td>9265.1</td><td></td></tr><tr><td>Albumin Fraction V</td><td>Carl Roth&amp;nbsp;</td><td>3854.2</td><td></td></tr><tr><td>THPC</td><td>Sigma Aldrich</td><td>404861-100ML</td><td>Toxic</td></tr><tr><td>0.1% Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ML</td><td>Slightly toxic</td></tr><tr><td>Phalloidin-rhodamine&amp;nbsp;</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>R415</td><td></td></tr><tr><td>3.7% Formaldehyde&amp;nbsp;</td><td>Life Technologies / Thermo Fisher&amp;nbsp;</td><td>F8775-25ML</td><td>Toxic</td></tr><tr><td>Rhodamine B</td><td>Sigma Aldrich</td><td>81-88-9</td><td></td></tr><tr><td>Filtropur S 0.2&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>2 83.1826.001&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>&amp;micro; slide 8 well</td><td>Ibidi GmbH</td><td>80826</td><td></td></tr><tr><td>KCSSGKSRGDS peptide</td><td>UPEP Ulm</td><td>Custom sysnthesis</td><td></td></tr><tr><td>Ethanol 99.8%, verg&amp;auml;llt</td><td>Carl Roth&amp;nbsp;</td><td>K928.5</td><td></td></tr><tr><td>Falcon 5 ml Polysterene Round-Bottom Tube&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>62.547.254&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Tubes 50 ml&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>62.547.254&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Tubes 1.5 ml&amp;nbsp;&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>72,690,001</td><td></td></tr><tr><td>Tubes 2 ml&amp;nbsp;&amp;nbsp;</td><td>Sarsted Ag und Co.</td><td>72,691</td><td></td></tr><tr><td>CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM</td><td>Greiner</td><td>655073</td><td></td></tr><tr><td>FreezeDryer Epsilon 1-6D,&amp;nbsp;</td><td>Christ, Osterode am Harz, Germany</td><td></td><td></td></tr><tr><td>Confocal Laser Scanning Microscope&amp;nbsp;</td><td>Carl Zeiss AG, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Zen Software Version 2012 Sp1, black edition, 407 version 8,1,0,484</td><td>Carl Zeiss AG, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>GSA Imaga Analyzer Software, GSA Image Analyzer, GSA, Version 419 3.8.7</td><td>GSA GmbH</td><td></td><td></td></tr></tbody></table>

easy manipulation of architectures in protein-based hydrogels for cell culture applications

ヒドロゲルは能力高い水和セル環境を提供するため細胞培養用有望な材料として認識されます。3 D テンプレートのフィールドは自然の細胞外マトリックスにそれらの材料の潜在的な類似性のため上昇しています。蛋白質ベースのゲルは、彼らは簡単に機能集積化することができます、調整可能な物理化学的性質と定義済みの構造を達成することが特に有望です。ただし、天然素材を使用して細胞培養用マクロポーラス 3 D テンプレートの生産はしばしば合成材料のそれらと比較して自分より弱い機械的性質によって制限されます。ここで、マクロポーラス ウシ血清アルブミン (BSA) を生成するさまざまな方法を行った-半径 10 を 70 μ m の範囲で調整可能な細孔径を持つハイドロゲル システムします。さらに、数百ミクロンの長いこのタンパク質ベース素材のチャンネルを生成する法を確立しました。膨潤率、pH、温度安定性、酵素分解挙動などの材料特性に及ぼす細孔径と同様に、毛穴を生成するさまざまな方法が分析されました。共焦点レーザ走査型顕微鏡を使用してゲルのネイティブ、腫れた状態で細孔径を調べた。タンパク質系の 2 つのモデル細胞系細胞接着 RGD ペプチド修飾を使用して細胞培養用可能性を評価した: ひと乳癌細胞 (A549) と adenocarcinomic ひと歯槽基底上皮細胞 (MCF7)。

Hydrogel development has become one of the most prominent fields in material research-related biological studies, with thousands of entries indexed in scientific research archives. Although the behavior of many systems is well studied, the manipulation of 3D networks, especially of sensitive protein-based materials, is often a major issue in material science. Another commonly underestimated challenge is the correct measurement of the native structure of a material using cryo electron micr...

Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

蛋白質ベースのヒドロゲルの三次元のアーキテクチャを操作するさまざまな方法は、材料特性に関してここで評価されます。マクロポーラス ネットワーク修飾が細胞接着ペプチドと細胞培養での実現性は、2 つの異なるモデルのセルラインを使用して評価されます。

細胞培養用ヒドロゲルの蛋白質ベースのアーキテクチャの簡単な操作

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

easy-manipulation-architectures-protein-based-hydrogels-for-cell

Research

JoVE Journal

Biochemistry

6.7K ビュー.  Ulm University. 蛋白質ベースのヒドロゲルの三次元のアーキテクチャを操作するさまざまな方法は、材料特性に関してここで評価されます。マクロポーラス ネットワーク修飾が細胞接着ペプチドと細胞培養での実現性は、2 つの異なるモデルのセルラインを使用して評価されます。

ビデオ: Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

機械的にダイナミックな細胞培養のための微細加工プラットフォーム

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

生物工学

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 ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

メカノキューの影響をデカップリングするためのヒドロキシのPAAmハイドロゲルの調製

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

マイクロパターンマクロスケール3D細胞構造のためのモジュラーヒドロゲルシートの構築

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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 ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生理活性タンパク質や生物学的応用の光化学ハイドロゲルのペプチドをパターニング

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

カプセル化と 3 D の細胞の免疫染色のエラスチン様タンパク質ゲルの生産

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

世界的な細胞のマトリックスメタロプロテアーゼと 3 D ゲルの代謝活性を測定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

ライブ顕微鏡イメージング下での3Dヒドロゲルの制御株

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...