著者は、彼らの思慮深い会話と材料科学の専門知識の教授ポール ・ ヴァン ・ タッセルと教授 Chinedum ・ オスジを感謝したいです。この仕事のための資金は、ライアンキャラハン新しいイニシアティブ賞、国立機関の健康 NIBIB BRPR01 EB16629 01A1 によって提供されました。

回使用し、すべての安全上の注意を使用する前すべての材料の材料安全データ シート (MSDS) をお読みください。
1. 酸化亜鉛針の合成
<ol>
	<li>0.04 M Zn (3)2の 250 ml * 6 H2O ソリューション硝酸亜鉛の 2.9749 g を 250 mL の水に追加することによって。</li>
	<li>6 g の水酸化ナトリウムを 150 mL の水に追加することによって 1 M NaOH の 150 mL を準備します。</li>
	<li>攪拌器、ホット プレートに鉱物油浴を設定し、常温で油浴に 500 mL の丸底フラスコを水没します。</li>
	<li>亜鉛 (3)2の 250 mL を追加 * 6 H2O、フラスコに反応を攪拌を開始。</li>
	<li>150 mL の NaOH 溶液 (白色沈殿物が簡単に形成および 2 つのソリューションがミックスするのにつれ消えてしまう) を追加します。発見 2 時間攪拌します。</li>
	<li>55-60 ° C に、フラスコの熱し、24 h の攪拌を続行します。</li>
	<li>熱をオフし、ソリューションを撹拌しながら室温に冷却します。</li>
	<li>11 μ m の細孔を Büchner 漏斗にソリューションをフィルター、乾燥させて一晩、覆いを取られました。フィルター ペーパーが注がれたソリューションから濡れているもはや漏斗穴上に白い粉が形成されたと、粒子は完全に乾燥させます。</li>
	<li>ZnO 針を収集し、針のような形態を確認する走査電子顕微鏡 (SEM) の準備します。簡単に、ピン スタブにカーボン テープをマウントし、ZnO 針をカーボン テープを中傷する金属のヘラを使用します。22.4 g/cm3の密度で 8 mm イリジウム コートをスパッタし、10 に画像を取得する kV。</li>
</ol>
2 HCl の顕微鏡のスライド犠牲亜鉛層の添加による PEG のリリース
<ol>
	<li>メタノール 200 mL に酢酸亜鉛の 1.756 g 溶解して酢酸亜鉛溶液を準備します。</li>
	<li>70% のエタノールと使い捨てワイプ 3"× 1" プレーン顕微鏡スライドをきれいに。乾いた空気で 10 分間乾燥させて。</li>
	<li>150 mm ガラス ホット プレート上にペトリ皿を置くし、150-160 ° C に予熱</li>
	<li>ピペット電球ガラス ピペットを使用して、酢酸亜鉛、スライド上に分散した薄層を形成の 5 滴を適用することでピンセットとコートのスライドとスライド ガラスを保持します。原液を滴下させる余分なソリューションを許可します。</li>
	<li>亜鉛アセテート コーティング面を上に向けて事前に加熱されたペトリ皿 (150-160 ° C) にスライドを配置します。15 分ホット プレートに残します。</li>
	<li>ピンセットでスライドを削除し、室温に冷却します。白の縞を持つコーティングするスライドが表示されます。</li>
	<li>使い捨て可能なワイプを使用して顕著な白い縞を削除するスライドの残りは任意の余分な亜鉛を削除します。表面を均一にする必要があります。</li>
	<li>公開、無菌性を確保するため、少なくとも 1 時間のためのバイオ セーフティ フードの光の紫外線に準備されたスライド。 注意: 紫外光は目に有害であるし、肌を露出しました。目や皮膚への直接露出を避けるため、使用していないときに電気供給をオフにします。</li>
</ol>
3. シリコーン アイソレータの作製
<ol>
	<li>正方形よりも小さい 1"× 1" にカット シリコン シート (アイソレータは 6 よく皿に収めることができるである必要があります)。</li>
	<li>生検パンチで正方形の中心に 8-12 mm の穴をパンチします。</li>
	<li>20 分間のオートクレーブ シリコーン絶縁 121.0 ° C および 1.12 kg/cm で。</li>
</ol>
4. PEG 溶液及びゲル合成のペグの準備
注: 機能化・重合 PEG の検討されている広範囲と前述の研究室や他の人によって。8,10,11,13,14,15,16,17
<ol>
	<li>RGD 細胞接着ペプチドを組み合わせてアクリロイル-ペグ-NHS 50 mM 炭酸水素ナトリウム (pH 8.5) のアクリル部分は、以前にされている生化学的反応とペプチドのアミン末端官能基化を有効にする 1:1 のモル比で効率 85% 以上を生成するために特徴付けられます。17</li>
	<li>300 mg/mL の濃度で 1-ビニル-2-ピロリドンと 2, 2-ジメトキシ-2-phenylacetophenone の混合光開始剤を準備します。</li>
	<li>1.5 mL チューブを別個で、以下重量を量り: ZnO 針 20 mg、ペグ-DA の 12.5 mg と 2.5 mg ペグ RGD の。</li>
	<li>270 μ 10 %fbs/PBS 溶液で酸化亜鉛針の 20 mg を中断します。ミックスする渦 (約 15 秒)。</li>
	<li>簡単に (&#60; 1 s) スピン チューブのベースに、ZnO 凝集体を持ってベンチトップ ミニ遠心分離機のソリューション。</li>
	<li>集計をチューブの底におく、ZnO ソリューションの上から 250 μ L を収集します。約 2.5 mm RGD 止め釘の解決を作成するペグ DA とペグ RGD と組み合わせます。ミックスする渦 (約 15 秒)。</li>
	<li>アセトフェノン/n ‐ ビニルピロリドン写真ハブと簡単にミックスする渦の 2 μ L を追加 (約 5 秒)。</li>
	<li>ZnO コーティング スライドの中央に沿って高分子溶液 20 μ L を追加します。</li>
	<li>ZnO コーティング面を上の 2 番目のスライドをゆっくりと下げるダウン (止め釘の解決は 2 つの ZnO 被覆層の間する必要があります)。空気の泡の形成を防止しようとします。任意の気泡を脱出し、薄い層にソリューションを普及するように主要な軸に沿って横にスライドを移動します。ポリマー溶液を使用してスライドの大部分をカバーします。後の手順でスライドを引き離すにことができることを確保するため一番上のスライドとわずかなオーバー ハングを作成します。</li>
	<li>架橋 15 分 365 nm の紫外線ランプ (約 10 mW/cm2) の下でスライド。</li>
</ol>
5. ペグのリリースがスライド ガラスからゲルします。
<ol>
	<li>張り出したスライドと手動でスライドを割ることを避ける 2 つのスライドを順番にゆっくりとしたペースで離れてプルに圧力を適用されます。少なくとも 5 分間乾燥一晩ゲルを許可します。</li>
	<li>滅菌 25 ミリメートルのガラス シャーレにスライドを配置し、優しくスライド (約 10-20 mL) をカバーする十分なだけを使用してスライドに 1 M 塩酸溶液を注ぐ。シャーレ; がみ合うゲルは、HCl 溶解亜鉛コーティングや針、スライドを持ち上げますが開始されます。ゲルはスライドから無料で、一度は、原液に HCl を注ぐ。</li>
	<li>スライドとゲルが水中に沈むまでゆっくりペトリ皿に約 25 mL の 1x PBS を注ぐことによって、ジェルを洗い流してください。</li></ol>慎重に PBS を廃棄物の容器に注ぐ。
	<li>ゲルがソリューションに浮かんでいるまで優しくペトリ皿に約 25 mL の 1x PBS を注ぐ。</li>
	<li>ピンセットを使用して、慎重にゲルの下シリコーン アイソレータを引き出して、それにゲルを持ち上げます。</li>
	<li>滅菌 1x PBS でいっぱい 6 よく皿にアイソレータとゲルを転送します。</li>
	<li>すべてのゲルのスライドから削除されており、PBS につかってまでこのプロセスを繰り返します。無菌性を確保するため、少なくとも 1 時間のためのバイオ セーフティ フードに UV ライトに準備ができてゲルを公開します。</li>

6. 細胞膜の播種
<ol>
	<li>A549 細胞の播種を備えます。簡単に言えば、5 mL の 1x PBS で 75 cm2フラスコを洗い流し、0.25% の 3 mL を加えるトリプシン-EDTA と 37 ° C で 2 〜 3 分に座ってみましょう<ol>
		<li>機械的にフラスコを扇動、10% 胎仔ウシ血清および 1% ペニシリン-ストレプトマイシン、3 mL ダルベッコ変更イーグル媒体との反応を抑制し、15 mL の三角フラスコに収集します。完全なダルベッコ変更イーグル メディアの追加 4 mL で洗い、同じ円錐形にまとめます。6 分の 475 x g でセルをスピンします。</li>
	</ol>
	</li>
	<li>セルを回転している中、6 ウェル プレートの各ウェルに完全なダルベッコ変更イーグル中の小滴を追加します。ゲルはメディアのドロップの休憩など、新しい 6 ウェル プレートにゲルにシリコーン アイソレータを転送するのにピンセットを使用します。今では、ゲルは、薄層に広がっている、目に見えない大きな穴がないことを確認します。これは直前の細胞播種乾燥ゲルの割れを避けるためにされるべきであります。</li>
	<li>10% 胎児牛血清および 1% ペニシリン-ストレプトマイシン 6 × 105セル/mL の濃度でダルベッコ変更イーグル培地で細胞を再懸濁します。パンチで抜いたシリコン膜部のメニスカスを維持しようとして、ゲルの中央に賢明な細胞懸濁液の滴を加えます。4 h の付着する細胞を許可します。</li>
	<li>4 h 後もダルベッコ変更イーグル完了の中の 0.5 mL を追加し、可能にする、完全密着の 37 ° C で一晩インキュベートします。、</li>
	<li>次の日に備える細胞播種 Huvec。<ol>
		<li>5 ml の 1x PBS の 75 cm2フラスコを洗い流し、0.25% の 3 mL を加えてトリプシン-EDTA と 37 ° C で 2 〜 3 分に座ってみましょう</li>
		<li>機械的にフラスコを扇動、3 mL 20% 牛胎児血清、1% ペニシリン-ストレプトマイシン、1% の成長サプリメントと M199 メディアとの反応を抑制し、15 mL の円錐形に収集します。</li>
		<li>M199 完全なメディアの 4 mL で洗い、同じ円錐形にまとめます。6 分の 475 x g でセルをスピンします。</li>
	</ol>
	</li>
	<li>下セルを回転している間は、新しい 6 ウェル プレートの各ウェルに完全培地 199 の小滴を追加します。シリコーンのセンターでメディアのドロップによく新しいシリコーン アイソレータを配置します。</li>
	<li>新しい 6 ウェル プレートのアイソレータに A549 細胞を PEG ゲルを支えるシリコーン アイソレータを反転慎重にピンセットを使用します。</li>
	<li>6 × 105セル/mL の濃度で比較を再懸濁します、シリコン膜の打ち抜き領域内のゲルのメニスカスを維持しようとして、賢明な反転ゲル ドロップの中央に細胞懸濁液を追加します。2 h の付着する細胞を許可します。</li>
	<li>2 時間後優しく M199 完全なメディアの 2 mL を各ウェルに追加します。</li>
</ol>
7. 蛍光抗体法
<ol>
	<li>慎重に手動ピペットとゲルからメディアを削除し、セルのシード、ゲルの中央に 4% パラホルムアルデヒド (PFA) の約 500 μ L を追加します。室温で 30 分間座ってみましょう。 注意: PFA は有毒な化学薬品;保護具を着用し、生物学的安全キャビネットで処理を行うようにします。</li>
	<li>慎重に PFA を削除し、PBS で 2 %bsa の約 500 μ L をゲルに追加します。室温で 1 時間放置します。</li>
	<li>BSA を慎重に取り外します。Pbs は、2 %bsa で 1: 100 希釈で一次抗体を準備、500 μ L をゲルに追加します。室温で 1 時間放置します。500 μ L の 1x PBS で優しくすすぎます。<ol>
		<li>二次抗体と同じプロセスを繰り返します。以下の抗体: 1) 抗ひと CD144 (VE カドヘリン) クローン 16B1;2) 抗ひと CD324 (E-カドヘリン) クローン 6714;3) 抗ひと CD31 (PECAM-1) クローン C-20;4) アンチ A549;5) 抗マウス FITC;6) Alexa Fluor 647 の反やぎ。F-アクチンを可視化、ファロイジン 500 μ L を追加 (PBS で 10 μ g/mL) 20 分。</li>
	</ol>
	</li>
	<li>慎重に二次抗体またはファロイジンを削除し、DAPI を 500 μ l 添加 (0.1 μ g/mL) 20 分の DAPI ソリューションを慎重に取り外し、ゲルが懸濁液で優しく各ウェルに 1.5 mL の 1x PBS を追加。<ol>
		<li>ピンセットやシリコーン絶縁を使用して、スライド ガラスに 1 つのゲルを転送します。イメージング、直前にゲルのとイメージング後 PBS ソリューションにゲルを交換します。</li>
		<li>また、DAPI マウント媒体を使用してゲルをマウントします。シールもマニキュアとサンプル、乾燥とゲルの割れを防ぐために 2 日以内の画像を取得します。トラブルシューティングには、表 1 を参照してください。</li>
	</ol>
	</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically-+and+mechanically-tunable+porated+polyethylene+glycol+gels+for+leukocyte+integrin+independent+and+dependent+chemotaxis.">Chemically- and mechanically-tunable porated polyethylene glycol gels for leukocyte integrin independent and dependent chemotaxis.</a> Technology. 02, 133-143 (2014).</li><li>DeLong, S. A., Moon, J. J., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+immobilized+gradients+of+bFGF+on+hydrogel+scaffolds+for+directed+cell+migration.">Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration.</a> Biomaterials. 26, 3227-3234 (2005).</li><li>Lauridsen, H. M., Gonzalez, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic,+ultrathin+and+elastic+hydrogels+regulate+human+neutrophil+extravasation+across+endothelial-pericyte+bilayers.">Biomimetic, ultrathin and elastic hydrogels regulate human neutrophil extravasation across endothelial-pericyte bilayers.</a> PLOS one. 12, 0171386-0171405 (2017).</li><li>Peters, E. B., Christoforou, N., Leong, K. W., Truskey, G. A., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(Ethylene+Glycol+Hydrogel+Scaffolds+Containing+Cell-Adhesive+and+Protease-Sensitive+Peptides+Support+Microvessel+Formation+by+Endothelial+Progenitor+Cells.">Poly(Ethylene Glycol Hydrogel Scaffolds Containing Cell-Adhesive and Protease-Sensitive Peptides Support Microvessel Formation by Endothelial Progenitor Cells.</a> Cellular and Molecular Bioengineering. 9, 38-54 (2016).</li><li>Schwartz, M. P., 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+synthetic+strategy+for+mimicking+the+extracellular+matrix+provides+new+insight+about+tumor+cell+migration.">A synthetic strategy for mimicking the extracellular matrix provides new insight about tumor cell migration.</a> Integr Biol (Camb). 2, 32-40 (2010).</li><li>Booth, A. J., 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=Acellular+normal+and+fibrotic+human+lung+matrices+as+a+culture+system+for+in+vitro+investigation.">Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation.</a> Am J Respir Crit Care Med. 186, 866-876 (2012).</li><li>Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membranes:+structure,+assembly+and+role+in+tumour+angiogenesis.">Basement membranes: structure, assembly and role in tumour angiogenesis.</a> Nat Rev Cancer. 3, 422-433 (2003).</li><li>Roudsari, L. C., Keffs, S. E., Witt, A. S., Gill, B. J., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+Poly(ethylene+glycol)-based+Tumor+Angiogenesis+Model+to+Study+the+Influence+of+Vascular+Cells+on+Lung+Tumor+Cell+Behavior.">A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior.</a> Scientific Reports. 6, 1-15 (2016).</li><li>Bermudez, L. E., Sangari, F. J., Kolonoski, P., Petrofsky, M., Goodman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+the+translocation+of+Mycobacterium+tuberculosis+across+a+bilayer+of+epithelial+and+endothelial+cells+as+a+model+of+the+alveolar+wall+is+a+consequence+of+transport+within+mononuclear+phagocytes+and+invasion+of+alveolar+epithelial+cells.">The efficiency of the translocation of Mycobacterium tuberculosis across a bilayer of epithelial and endothelial cells as a model of the alveolar wall is a consequence of transport within mononuclear phagocytes and invasion of alveolar epithelial cells.</a> Infect Immun. 70, 140-146 (2002).</li></ol>

著者が明らかに何もありません。

ここで詳細なプロトコルをバイオミメティック BM 足場として機能する可変ペグ ハイドロゲルを作成できました。具体的には、さまざまな PEG 分子量、ペプチドの活用戦略と酸化亜鉛微結晶構造や濃度、弾性率、生化学的性質と、ヒドロゲルの細孔構造変更できます、それぞれ。極薄のペグ足場は高い気孔密度と基底膜標準透過性のサポート モデル (表 2) と比較して体内で...

細胞外マトリックス (ECM) は細胞接着をサポートし種類の異なる細胞の間の障壁として機能および複雑な組織や臓器の重要なコンポーネントは、タンパク質の足場を作る。間質結合織と対照をなして基底膜 (BM) は互いから組織のコンパートメントに分割するバリアとして機能する ECM の特殊な型です。BMs は約 100 μ m、厚さ、従ってどちらかの側の細胞間の直接的および間接的な通信を可能にします。BMs の 2 つの一般的な例は、ペリサイトおよび内皮細胞間の血管壁は、血管の BMs と内皮細胞および上皮細胞の間にある気道 BMs です。BMs は役立つ細胞極性や移行などの細胞機能の調節に、健康および病気の重要な役割です。1構成、剛性、アーキテクチャ、および BMs の気孔率は、異なる生理機能を容易にする器官システムによって異なります。たとえば、BM 毛穴は感染時に炎症や細菌中の免疫細胞の移動、細胞間コミュニケーション、可溶性分子拡散を維持するために重要です。気道で毛穴は直径 0.75 から 3.86 μ m2に至るまでの BM の完全な厚さにまたがる
BM の薄い性質が細胞の種類は、1 つの別から物理的に分離、パラクリンと連絡先を介したシグナルを介した細胞間コミュニケーションが維持されることを保証します。したがって、人間の病気のin vitro での研究に研究者は培養細胞膜に多孔質の透水性サポート挿入に頼ってきました。3これらのモデルは、重要な健康および病気の役割を果たしている細胞のコミュニケーションを理解するためにされています。3,4,5,6,7透水性サポート挿入白血球動員や細菌浸潤; などの生理学的プロセスを調節する細胞シグナリング方法を理解するための基本的な要件を満たすただし、挿入の重要な制限があります、人間点換気サポートを模倣する失敗挿入機械的および生化学的な可変性がないし、単純な多孔質構造が不規則な毛穴を作成する線維状構造を模倣していませんBMs の典型的です。したがって、細胞プロセスに影響を与える固有の BM プロパティを再作成することができます可変システムの成長の必要性があります。
ポリマー ベースの基板は、バイオミメティック体内環境をもっと密接に模倣するコンテキストで細胞膜を研究する BMs の開発のための理想的な候補です。8,9,10,11,12ポリマーが機械的に可変、バイオミメティック ペプチド断片.を組み込むこと化学的に変更することができます。11,12,13不活性ポリマー, ポリエチレング リコール (PEG) バイオミメティック BMs を作成する使用することができます、最近の作品の詳細な細胞の成長をサポートする多孔質ネットワークと機械的に可変のペグ アルギニン-グリシン-アスパラギン酸 (RGD) ゲルの合成・炎症細胞走化性。14公開ペグ ベースが基板は、透過性のサポートする範囲よりヒトの ECM のより現実的なモデルを提供、これらのモデルの多くは細胞膜の文化を作成する能力を制限する約 775 μ m の深さを持つ、非常に厚い、連絡先。14
現在の細胞膜培養技術の制約の多くを克服するペグのポリマー ベース BM 模倣を作成するためのプロトコルを紹介します。ポリマーの合成と架橋は、その後、選択的にから削除中に酸化亜鉛微結晶生産の製造に広く使用された材料を組み込んだテンプレート メソッドを開発している、結果として得られるバルク高分子。このプロセスは、人間の BMs の蛇行と相互接続の細孔ネットワークを模倣したランダム多孔性ネットワークを生成します。さらに、気孔率はサイズと針生産時に反応の化学量論の変更を介して酸化亜鉛微結晶の形状を変更することによって変更できます。ここで開発した技術は人間の点最後に、力学、気孔率、厚さを模した超薄ハイドロゲルを作成し、これらの BM のような構造の生化学的組成はほとんどは微小環境を生成する簡単に変更できます。その見られる生体内に似ています。

<table border="0" cellpadding="0" cellspacing="0" style="width:737px;" width="738">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1M Hydrogel Chloride (HCl)</td>
			<td style="width:117px;">EMD</td>
			<td style="width:119px;">HX0603-75 2.5L</td>
			<td style="width:167px;">Sterile. Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">1X PBS</td>
			<td style="width:117px;">Gibco</td>
			<td style="width:119px;">14040-133 500 mL</td>
			<td>None</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">Zinc Nitrate Hexahydrate (Zn(NO3)2&#8226;6H2O)</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">228737-500g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Sodium Hydroxide (NaOH)</td>
			<td style="width:117px;">Macron Chemicals</td>
			<td style="width:119px;">278408-500g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:335px;">Zinc Acetate Dihydrate ((CH3O2)2Zn2+&#8226;2H2O)</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:119px;">AC45180010 1 kg</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">Methanol (CH3OH)</td>
			<td style="width:117px;">J.T. Baker</td>
			<td style="width:119px;">9070-05 4L</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">VWR Life Science Seradigm Premium Grade FBS</td>
			<td style="width:117px;">VWR</td>
			<td style="width:119px;">97068-085</td>
			<td>Sterile filter. 5 mL FBS in 45 mL PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Mineral oil</td>
			<td style="width:117px;">CVS&#160;</td>
			<td style="width:119px;">PLD-B280B</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Round bottom flask</td>
			<td style="width:117px;">ChemGlass</td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Thermometer</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Stir bar</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Plain precleaned microscope slides 3&#34;x1&#34;x1&#34; mm thick</td>
			<td style="width:117px;">Thermo Scientific</td>
			<td style="width:119px;">420-004T</td>
			<td>Spray with ethanol and let dry prior to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass pasteur pipets</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1 mL rubber bulbs</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Plastic 100 mm petri dishes</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Sterile forceps</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Silicone isolators</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>0.8 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Polydimethylsiloxane (PDMS) punches</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass bottles</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">6 well plates</td>
			<td style="width:117px;">Cellstar</td>
			<td style="width:119px;">657 160</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Filter Paper</td>
			<td style="width:117px;">Whatman</td>
			<td align="right" style="width:119px;">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Stirrer-hot plate</td>
			<td style="width:117px;">VWR Dya-Dual</td>
			<td style="width:119px;">12620-970</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:335px;">2,2-Dimethoxy-2-phenylacetophenone (C6H5COC(OCH3)2C6H5</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">24650-42-8</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-Vinyl-2-pyrrolidone (C6H9NO)</td>
			<td style="width:117px;">Aldrich</td>
			<td style="width:119px;"></td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:335px;">Polyethylene Glycol 10,000 (H(OCH2CH2)10,000OH)</td>
			<td style="width:117px;">Fluka</td>
			<td style="width:119px;">81280-1kg</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">RGDS</td>
			<td style="width:117px;">Life Tein</td>
			<td align="right" style="width:119px;">180190</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Blak-Ray long wave UV lamp</td>
			<td style="width:117px;">UVP</td>
			<td style="width:119px;">Model B 100AP</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Eppendorf tubes</td>
			<td style="width:117px;">USA Scientific</td>
			<td style="width:119px;">1615-5500</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>

ultrathin porated elastic hydrogels as a biomimetic basement membrane for dual cell culture

基底膜は、剛性、組成、アーキテクチャ、および気孔率で変わることができる細胞膜の重要なコンポーネントです。内皮細胞上皮膜のin vitro研究伝統的二層文化を有効にすると透過性のサポート モデルに依存しているが、透過性のサポート、人間の基底膜の多様性を複製する能力に制限が。対照的に、化学合成を必要とするハイドロゲル モデル高調整可能、素材の剛性とバイオミメティック ペプチッドまたは蛋白質の定款による生化学的組成の両方の変更を可能にします。ただし、従来のハイドロゲル モデルは、細胞間の接触と移民研究機能の in vitroの毛穴がないため機能に制限されます。さらに、伝統的なゲルの厚さによるハイドロゲルの全体の厚さにまたがる毛穴定款は挑戦されています。本研究で我々 は生体模倣ヒドロゲルの以前の欠点に対処するのに poly-(ethylene-glycol) (PEG) ゲルおよび亜鉛酸化物テンプレート メソッドを使用します。その結果、可変間隙のアーキテクチャ、機械的性質、成分とカスタマイズ可能な足場にコンフルエントの細胞膜の文化を可能にする極薄、基底膜のようなハイドロゲルを提案します。

ペグ RGD ヒドロゲルは、2 つの犠牲酸化亜鉛層間高分子溶液を挟むと酸化亜鉛の針で毛穴テンプレートの作成によって形成されました。いけにえの亜鉛酸化物は塩酸、連続気孔 (図 1) と極薄のペグ ゲルを生成し取り外しました。酸化亜鉛針の形態を走査型電子顕微鏡 (SEM)、確認したし、それぞれ 3.92 ± 0.089 μ m および 0.43 ± 0.02 μ m にする平均の?...

Watch this Scientific Journal Video about Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture at JoVE.com

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

現在の二層文化モデルは模倣する生体内微小機能の in vitro研究できないようにします。ポリエチレング リコール、酸化亜鉛テンプレート メソッドを使用してこのプロトコル記述する可変剛性、気孔率、体内によく似た成分と、極薄のバイオミメティック基底膜の開発細胞外マトリックス。

デュアルのバイオミメティック基底膜として極薄 Porated 弾性ゲルの細胞培養

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

ultrathin-porated-elastic-hydrogels-as-biomimetic-basement-membrane

Research

JoVE Journal

Bioengineering

7.7K ビュー.  Yale University. 現在の二層文化モデルは模倣する生体内微小機能の in vitro研究できないようにします。ポリエチレング リコール、酸化亜鉛テンプレート メソッドを使用してこのプロトコル記述する可変剛性、気孔率、体内によく似た成分と、極薄のバイオミメティック基底膜の開発細胞外マトリックス。

ビデオ: Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巣と卵管の三次元器官培養用アルギン酸ゲル

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

アクティブな細胞培養のための形状記憶ポリマー

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三次元バイオミメティック・テクノロジー：小説Biorubberはコラーゲンハイドロゲルで定義されたマイクロ・マクロスケールのアーキテクチャを作成します。

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

治療用途のためのツールとしてのRGD官能ヒドロゲルの合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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 Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水質浄化のための膜のような防汚特性を持つハイドロゲルの合成

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

ヒアルロン酸を用いた神経膠芽腫患者由来細胞の 3次元培養ヒドロゲル

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...