1。皮膚外植片からの線維芽細胞培養の確立<ol><li> 100単位/ mlペニシリン、100μg/ mlのストレプトマイシン、及び0.25μg/ mlのファンギゾンを補った人間の前腕から取得した4ミリメートルパンチ生検をMEMに配置されます。 </li><li>任意の皮下脂肪をトリミングして、細かくシャーレの底面に24番メス刃を揺動することにより、小さな断片に生検を切り刻む。 </li><li>フラスコの表面になるまで一次性線維芽細胞増殖培地（MEM、10％FCS、100単位/ mlペニシリン、100μg/ mlのストレプトマイシン、及び25μg/ mlのファンギゾンを補充）を25cm 2の組織培養フラスコに入れ、組織のスラリーは、追加された覆われたが、液体の深さは、組織が浮遊するのは不十分である。 </li><li> 5％CO 2の加湿雰囲気中で37℃で3日間インキュベート℃に続いて、プライマリー繊維芽細胞増殖培地3mlを加える。 </li><li>新鮮なプライマリー繊維芽細胞増殖培地を使用して別の3日間のインキュベーション変化媒体の後、またはCEL場合lsはほぼコンフルエントになる彼らは、新鮮な培地に1:4に分割されることがあります。 （ファンギゾンはこれ以降省略できます）。 </li></ol> 2。ステージI - ラットの尾からコラーゲンIの調製注：約12から14思春期（生鮮または冷凍）ラットの尾のためのプロトコル<ol><li> 70％エタノールで洗浄することにより、ラットの尾を用意し、次のように腱を削除します。 </li><li>尾の長さに沿ってメスをむきで上から下へ尾の真ん中にスライスして、ラットの尾部から皮膚を取り除きます。 </li><li>尾の近位領域のコアから腱をデタッチします。 </li><li>歯鉗子を用いてシースを避け尾の先端領域に向かって腱を削除します。 </li><li> 48時間4℃で撹拌することにより腱の1グラム/ 0.5 M酢酸250mlを抽出します。 </li><li> 30分間抽出物（7,500×gで）を遠心し、ペレットを捨てる。 </li><li>上清にNaClを10％（w / v）の等量を追加し、30〜60分間かき混ぜる。 </li><l30分のi&gt;遠心分離（10,000×g）で、4℃で24時間撹拌した〜1:1の比率で上清を捨て、0.25 M酢酸で再溶解沈殿℃に</li><li> 6Lの6-8変化〜17.5 mM酢酸（冷たい水のリットル当たり1ミリリットル氷酢酸、変化2×毎日）に対してコラーゲン溶液を透析する。 </li><li> 1.5時間、30,000 xgで透析コラーゲンを遠心分離します。 </li><li>無菌フラスコ内の上澄みと場所を削除します。 </li><li> 〜2 mg / mlの濃度に私は4で0.5mMの酢酸を用い℃のコラーゲンを調整</li></ol> 3。ステージII - 埋め込まれた繊維芽細胞（8日間の契約を許可する）と3Dマトリックスを設定します。 <ol><li> 4時に冷たい試薬を使用してミックスを組み立てる°予冷ボトルのC。氷の上でコラーゲンIを保管してください。 </li><li>コンフルエントプライマリー繊維芽細胞の1 T75フラスコ（〜細胞数1×10 6）のために使用： <ul><li> 25mlのラット尾部コラーゲン（約2mg/mLの濃） </li><li> 10xの3ミリリットルMEM </li><li> 0.22 M NaOHを：まず最初に、コラーゲンがオレンジ色に点​​灯するまで、攪拌2ミリリットル、次に滴下しながら追加し、 </li><li>はなく、ピンク（通常最大3ミリリットル）。おおよそのP H = 7.2。 </li><li>注：これは、少しでもアルカリ性条件にさらされると線維芽細胞がゲルにかからないように培地/ゲルは中性またはわずかに酸性のままであることを確認することが重要です。 </li></ul></li><li> 、線維芽細胞をTrypsinise ​​5分間400 xgでスピンし、上清を除去します。 </li><li> 5分の間に、上記スピン：12×35ミリメートルプラスチック皿を準備する - 通常は線維芽細胞/コラーゲンミックスの1つのフラスコから12皿を達成。 </li><li> 3ミリリットルFCSで線維芽細胞を再懸濁するとすぐにコラーゲンミックスに追加し、かき混ぜる。 </li><li>プレートの約2.5ミリリットル皿当たりコラーゲン/線維芽細胞、できるだけ早く。気泡やコラーゲンの設定を避けるようにしてください。 </li><li>コラーゲンは10分間空気中5％CO 2の加湿雰囲気中で37℃で設定してみましょう。 </li><li>成長する線維芽細胞の1ミリリットルを追加目の培地（DMEM +10％FCS）。 </li><li>ピペットを用いて皿の両側からコラーゲン/線維芽細胞マトリックスをデタッチします。 </li><li>次の日：繊維芽細胞増殖培地（DMEM +10％FCS）の1ミリリットルを追加します。 </li><li>一日おきにメディアを変更してください。彼らは、24ウェルディッシュに収まるまで、約8日間の契約にコラーゲン/線維芽細胞マトリックスを許可する（直径1.5cmに〜3.5 cmから収縮、図1を参照）。 </li></ol>注：コラーゲン濃度は、アプリケーションに合わせて調整する必要があります。より多くのコラーゲン溶液を希釈し、速く、それは線維芽細胞によって収縮されます。収縮率のわずかな違いは、同じ濃度でコラーゲンの異なるバッチで経験されます。同様に、種々の線維芽細胞培養は、収縮速度より速い、異なるレートであり、本より線維芽細胞のコラーゲンゲル収縮する。コラーゲン濃度および線維芽細胞密度が故にによって収縮率を変更するために調整することができ、コラーゲンゲルの、最終的な密度。 4。ステージII - マトリックスの上に興味のあるメッキ細胞注意：使用前にエタノールですべての鉗子や機器を滅菌する。 <ol><li>鈍鉗子を使用して、静かに24ウェルディッシュに委託行列を移動します。それが倍にしないことを確認してください。 </li><li>マトリックスの上に約4×10 4個 / mlとプレート1ミリリットル（トリプシンを取り除くためtrypsinising、スピン細胞の後）に興味のある細胞の懸濁液を準備します。必要なセルの実際の数は、使用する細胞の種類によって異なります。細胞培地は、目的の細胞の正常な増殖培地である必要があります。 </li><li>細胞は約3〜5日、マトリックスの上に合流に成長することができます。 </li></ol> 5。ステージIII - 侵略のためのグリッド（約0から21日）に行列を転送<ol><li>三脚を作成します（図2を参照してください）​​使用前にオートクレーブにステンレス鋼の格子を切った。 </li><li>に滅菌グリッドを配置6cmディッシュには、グリッド上のレベル（約10.5ミリリットル）に増殖培地を追加します。場所は、グリッド上の行列と行列の底がメディアに接触しているが、水没しないように静かに培地を吸引。これは、侵略を推進してグラデーションを作成し、空気/液体界面、と呼ばれています。 （図2を参照）は2日ごとに培地を交換してください。 </li><li>細胞を生き、蛍光細胞を用いた文脈イメージングは​​、この段階またはそれ以前のバージョンで画像化することができる。委託または線維性コラーゲンは、私は第二高調波発生（SHG、図3を参照）を使用して画像化することができる。我々は細胞質GFPを発現する細胞は、少なくとも200μmの深い撮像することができる一方、SHGは、マトリックス内に少なくとも100μmを撮像することができることがわかり、広い視野検出と組み合わせた多光子励起を用いた。 </li></ol>注：浸潤の定量化に関して、matriciesがグリッド上に配置された日を0日を定義します。グリッド上の配置は、侵略のintを促進する細胞培養培地のグラデーションを生成<html>行列をO。 （参考文献一覧表を参照）などの侵入、増殖、生存や分化などの生物学的プロセスを評価するために、21日（またはそれ以上） - サンプルは、次の1の上に結像させることができる。 6。ステージIV - ギャグ<ol><li>ファルコンチューブに4％PFAの5ミリリットルを追加します。 </li><li>平らな表面に行列を転送します。新鮮なきれいなメス（あなたは断面を染色されるように）と半分に行列を切り取り、一晩固定し、メス、4％PFAへの転送で行列を持ち上げる。 </li><li>器官の行列になりました（図3参照）選択抗体/染色で染色する準備が整いました。 </li></ol> 7。代表の結果： <img alt="図1" src="/files/ftp_upload/3089/3089fig1.jpg" /> 図線維芽細胞とシャーレから切り離されて6-8日以上の契約を許可ラット尾部コラーゲンの線維芽細胞の契約コラーゲンIの混合物の1例 。 図2は &quot;src =&quot; / files/ftp_upload/3089/3089fig2.jpg &quot;/&gt; 2器官アッセイの進行を示しています。セットアップ、器官の概略と進行B、ステンレス鋼の上にコラーゲン/線維芽細胞マトリックスの例は、空気/液体界面を作成するための無菌グリッド。 <img alt="図3" src="/files/ftp_upload/3089/3089fig3.jpg" /> 器官型アッセイの3アプリケーションを示しています。 A、B、非侵襲的および侵襲性膵管腺癌（PDAC）細胞が器官行列で時間をかけて侵入C、線維芽細胞、契約コラーゲン真皮成分で層状表皮を示す皮膚等価物を生きD、線維芽細胞に侵入C8161メラノーマ細胞はコラーゲン真皮同等E、器官マトリックス内に相互作用し、線維芽細胞（赤）と侵略侵襲PDAC細胞（緑）縮小した。Fは </stronグラム&gt;、分解周囲の細胞外マトリックスとの相互作用侵襲PDAC細胞（緑）（紫）多ベースの第二高調波発生を用いて可視化した。

<ol>
	<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li><li>Edward, M., Gillan, C., Micha, D., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+regulation+of+fibroblast+hyaluronan+expression:+a+mechanism+to+facilitate+tumour+growth+and+invasion.">Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion.</a> Carcinogenesis. 26, 1215-1223 (2005).</li><li>Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+breast+cancer+cells+in+vivo+requires+both+paracrine+and+autocrine+loops+involving+the+colony-stimulating+factor-1+receptor.">Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.</a> Cancer Res. 69, 9498-9506 (2009).</li><li>Serrels, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+study+of+E-cadherin+and+membrane+dynamics+in+living+animals:+implications+for+disease+modeling+and+drug+development.">Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.</a> Cancer Res. 69, 2714-2719 (2009).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li><li>Edward, M., Quinn, J. A., Pasonen-Seppanen, S. M., McCann, B. A., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Methylumbelliferone+inhibits+tumour+cell+growth+and+the+activation+of+stromal+hyaluronan+synthesis+by+melanoma+cell-derived+factors.">4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.</a> Br. J. Dermatol. 162, 1224-1232 (2010).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+characteristics+of+transformed+keratinocytes+from+mouse+and+human+skin+in+vitro+and+in+vivo.">Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.</a> J. Invest. Dermatol. 81, 168s-175s (1983).</li><li>Nystrom, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J. Pathol. 205, 468-475 (2005).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J. Cell. Biol. 185, 11-19 (2009).</li><li>Amjad, S. B., Carachi, R., Edward, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+regulation+of+TGF-beta+and+connective+tissue+growth+factor+expression:+a+role+in+suppression+of+scar+tissue+formation.">Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.</a> Wound Repair Regen. 15, 748-755 (2007).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nat. Cell. Biol. 9, 1392-1400 (2007).</li></ol>

我々は、開示することは何もない。

ここでは、侵襲的な細胞遊走6-8の研究に適した3次元マトリックスの産生するための方法を提示する。行列は初代ヒト表皮繊維芽細胞によって数日間にわたって契約しているコラーゲンタイプ1線維で構成されています。コラーゲンは、ポリペプチドの両端に反応性を保持し、バッファー条件9を中和する際に大きな凝集体にコラーゲン線維の架橋を促進する酸抽出ではなく、酵素消化することにより調製される。より密接に、生体内でのコラーゲンの機能を模倣し、ゲル中で培養した細胞の浸潤特性にとって重要な意味を持って再構成し、ゲル中のこの結果。コラーゲンの架橋は、若い動物ではより不安定であるので、それが新鮮または凍結出発物質が使用されているかどうかは重要ですが、思春期のラットの尾を使用しないことが重要です。若い動物からコラーゲンIはw従って抽出することが容易であり、再構成i番目に高い忠実度。 コラーゲンマトリックスを固定し、染色し浸潤性腫瘍細胞又は間質線維芽細胞2,10のいずれかに対して向けられた抗体を用いて5,6および浸潤を抑制する可能性がある薬剤の有効性をテストするために非常に有用であることができます。 このプロトコルは、初代ヒト皮膚線維芽細胞の使用を示唆しているが、それは調査対象組織の種類に応じて、他の組織型からプライマリー繊維芽細胞を使用することが可能である。それが安定して蛍光タンパク質複合体でトランスフェクトされた細胞を含む不死化線維芽細胞を用いて、文化を確立することも可能です。このように両方のストローマ細胞と腫瘍細胞が浸潤11時の細胞間相互作用を可視化するためにラベルを付けることができます。

<table border="1"><tbody><tr><th>試薬の名称</th><th>会社</th><th>カタログ番号</th><th>コメント（オプション） </th></tr><tr><td> 10xのMEM </td><td>ギブコ</td><td> 21430 </td><td> - </td></tr><tr><td> NaOHを</td><td>シグマ</td><td> 367176-500G </td><td>水に0.22 Mのストックを準備</td></tr><tr><td> FCS </td><td> PAAラボラトリーズ</td><td> A15-101 </td><td> - </td></tr><tr><td> 35ミリメートル皿</td><td>ファルコン</td><td> 353001 </td><td>ステップ3.4の</td></tr><tr><td> 60ミリメートル皿</td><td>ファルコン</td><td> 353004 </td><td>ステップ5.2の</td></tr><tr><td>春鉗子鈍</td><td>サムコ</td><td> E003/02 </td><td>スムーズではありません、歯</td></tr><tr><td> 16％パラホルムアルデヒド</td><td>電子顕微鏡のサービス</td><td> 15710 </td><td>使用前にPBS中の4％に希釈</td></tr><tr><td> CD-1、サイズ40メッシュのスクリーン</td><td>シグマ</td><td> S07707-5EA </td><td>ステンレススチールグリッド、パックごとに5 </td></tr><tr><td>透析チューブ</td><td> Medicell国際</td><td> 7607 2295 </td><td> 12から14 kDの</td></tr><tr><td> PBS </td><td>オキソイド</td><td> BR0014G </td><td> - </td></tr><tr><td>酢酸</td><td>シグマ</td><td> 242853 </td><td> - </td></tr><tr><td> 24ウェルディッシュ</td><td>ファルコン</td><td> 353047 </td><td>ステップ4.1の</td></tr><tr><td>ファンギゾン</td><td> vitrogenで</td><td> 15290018 </td><td> - </td></tr></tbody></table>

organotypic collagen i assay: a malleable platform to assess cell behaviour in a 3-dimensional context

細胞遊走は、開発、創傷治癒、免疫系の細胞応答、腫瘍細胞の転移を含む生物学の多くの側面に基本的である。移行は、光学顕微鏡で調査に従順セルラーダイナミクスを作るためにガラスカバースリップ上に研究されてきた。しかし、これは細胞遊走の多くの側面を忠実にガラスやプラスチックなどの硬質1二次元の基板では表現されないため、その弾力性、タンパク質組成物、および細孔径など、ローカル環境の機能に依存することが明らかになってきた。さらに、間質線維芽細胞2、免疫細胞3を含む他の細胞型との相互作用は、癌細胞の浸潤を促進する上で重要な役割を果たすことが示されている。分子レベルでの調査がますます比較した場合、同一の細胞の薬物治療への反応を含む分子動力学は、大きく異なっていることが示されている<em&gt; in vitro および in vivoの4インチ 理想的には、生体内でその天然に存在する文脈における細胞遊走を勉強するのがベストですが、これは常に可能とは限りません。そのような細胞由来マトリックス、マトリゲル、器官培養（ここで説明）組織外植片、オルガノイド、および異種移植片のような中間組織培養システムは、それゆえに重要な実験的な中間体である。これらのシステムは生体内環境でのおおよその特定の側面を、そのような安定的にトランスフェクトされた細胞株、薬物治療計画、長期的かつ高分解能イメージングの使用などの実験操作に対してより適している。このような中間システムは 、in vivo で 5イメージングを引き受ける前にプローブを検証し、イメージに細胞や蛍光レポーターの動的応答に必要なパラメータを確立するための根拠を証明として特に有用である。このように、彼らは、リヴィンの実験の必要性を低減する上で重要な役割を果たすことができるG動物。

Watch this Scientific Journal Video about Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context at JoVE.com

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

メソッドは、I型コラーゲンと初代ヒト線維芽細胞からなる3次元マトリックスの調製について記載されています。この器官ゲルは、それが組織の間質の基本的な機能を模倣し、顕微鏡の多くの形態が可能であるため、侵襲的な細胞遊走を評価するために有用な基質として機能します。

器官型コラーゲンIアッセイ：3次元コンテキストでのセルの挙動を評価するための可鍛性プラットフォーム

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

organotypic-collagen-i-assay-malleable-platform-to-assess-cell

Research

JoVE Journal

Bioengineering

22.3K ビュー.  University of Glasgow. メソッドは、I型コラーゲンと初代ヒト線維芽細胞からなる3次元マトリックスの調製について記載されています。この器官ゲルは、それが組織の間質の基本的な機能を模倣し、顕微鏡の多くの形態が可能であるため、侵襲的な細胞遊走を評価するために有用な基質として機能します。

ビデオ: Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

タンパク質分解的に分解性3次元システムにおける卵胞のカプセル化と文化のための方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

生物工学

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3次元自己組織化ペプチドハイドロゲル中のヒト神経前駆細胞の栽培

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

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

3次元細胞培養のための自己申告足場

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

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

皮膚からのI型コラーゲンの調製のための改良された方法

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

3Dコラーゲンマトリックス中の走化性を調査するために、平面グラデーション拡散システム

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

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

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

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

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

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

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

組換えコラーゲン ペプチド Microcarriers 細胞伸長と細胞バイオリアクター配信システムとして潜在的な使用のためのモデルします。

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.