The authors thank the Fraunhofer ISC for the collaboration concerning the construction of the automated wounding device. The project was founded by Fraunhofer internal project &#8220;M&#228;rkte von &#220;bermorgen&#8221; (SkinHeal).

注：プロトコルは24全層皮膚同等物の生産のために設計されている。ヒト皮膚線維芽細胞と表皮角化細胞は、以前に公開されたプロトコルに従って、皮膚生検から単離した。21,22インフォームドコンセントが事前に得られたものであり、研究はユリウスマクシミリアン大学ヴュルツブルクの人間研究に制度的倫理委員会によって承認された（182投票/ 10）。 皮膚コンポーネントの1。生産<ol><li>を6mg / mlの最終濃度まで0.1％酢酸を有するコラーゲンを溶解する。ゲル中和溶液は、232.5ミリリットル2回DMEM、7.5ミリリットルのウシ胎児血清、7.5ミリリットル3 M HEPES、2.5ミリリットルコンドロイチン硫酸（5ミリグラム/ ml）を混合する。氷の上でゲル中和溶液とコラーゲン溶液を保管してください。挿入/肌当量当たりゲルの500μlのを計算します。 NOTE：ゲル中和溶液は、コラーゲン溶液のprepaの2つの部分と混合されるようにコラーゲンの倍の量の再。 </li><li> 24ウェルプレートに24のインサートを置きます。 </li><li> 10ミリリットルDMEM、270×gで5分間遠心分離器でヒト皮膚線維芽細胞（HDF）を再懸濁し、細胞を数える。 G X 270で5分間に必要なHDFの量（500μlのコラーゲンゲルあたり5×10 4細胞）と遠心細胞を抽出します。 </li><li>上清を除去し、慎重に気泡を発生させずに、ゲル中和液を冷却して4ミリリットルでHDFを懸濁します。このステップから、コラーゲンゲルの早期凝固を避けるために、可能な限り高速に動作することを確認してください。 </li><li>コラーゲン溶液8mlの溶液を再懸濁する。 </li><li>マルチステップ·ピペットでコラーゲン - 細胞混合物を取り、各挿入で急速混合物500μlのを埋める。ゲルをゼリー状にするために、インキュベーター（37℃、5％CO 2）中で20分間ゲルをインキュベートする。 </li><li>ウェル当たり2ミリリットルダルベッコ改変イーグル培地（DMEM）+ 10％ウシ胎児血清（FCS）でゲルを沈めるインキュベーター（37℃、5％CO 2）で24時間静置する。 </li></ol>ヒト表皮ケラチノサイト（HEK）2.追加<ol><li> 24時間後に培地を除去します。を50μg/ mlの最終濃度になるように超純水でフィブロネクチンヒトタンパ​​ク質を溶解する。フィブロネクチン溶液25μlの各真皮同等物をカバーしています。インキュベーター（37℃、5％CO 2）中で30分間ゲルをインキュベートする。 </li><li>その間、10mlのKGM-2でHEKを再懸濁し、KGM-2 5 +準備％FCSで1×10 6細胞/ mlに細胞濃度を設定する。各ゲル上の種子1×10 5 HEK。細胞が付着できるように、インキュベーター（37℃、5％CO 2）中で45分間ゲルをインキュベートする。 </li><li>インキュベーター（37℃、5％CO 2）中で、5％FCS +準備し、培養それらKGM-2を有するゲルを沈める。 </li></ol>全層皮膚等価物の3.文化（FTSE） <ol><li> 6-7日間培養しFTSEFCS濃度（5％、2％、および0％FCS）降順。次に低いFCS濃度が2〜3日ごとに培地を変更します。このために、完全に培地を除去し、新鮮な培地の1.6ミリリットルを追加します。 </li><li> 7日目に、完全培地を除去。 注：そうしながら、ピペットの先端でFTSE表面に触れないでください。 </li><li>滅菌ピンセットで6ウェルプレートの1ウェルに挿入するたびに配置します。 FTSE表面を濡らしていないことを確認、ウェル当たり1.5ミリリットル空輸媒体を追加します。さらに14日間、2〜3日ごとに培地を変更します。 注：メディアの充填レベルは、FTSEのメニスカスまでです。 </li></ol> 4.組織学的分析<ol><li>固定し、FTSEのパラフィン包埋<ol><li>培地を除去、新鮮な24ウェルプレートにインサートを転送し、各ウェルに、4％パラホルムアルデヒド、1.6を加えて組織を固定し、室温で2時間、組織をインキュベートする。注意！パラホルムアルデヒドは、ヒュームフードの下で操作し、有毒である。転送組織埋め込みカセットにFTSE。水で洗浄することにより、残りの固定液を除去し、昇順エタノール濃度を用いて組織を脱水。 </li><li>真ん中の縦のカットによる皮膚同等物を分割します。下向きに切断面を持つパラフィン充填金属ベース金型に2枚を配置します。バッキングとして金型の上に組織カセットを追加します。 </li><li>適切な顕微鏡スライド上にスライドマウント、3-5程度のスライスをカットし、straitening、40℃の水浴上でそれらをフロート。徹底的に乾燥スライス。 </li></ol></li><li>ヘマトキシリン​​＆エオシン（H＆E）および免疫組織化学（IHC）染色<ol><li>場所は、ラックに挿入し、60℃で1時間インキュベートする。パラフィンが溶融していることを確認します。ステインは、標準化された概要染色などのH＆Eとのスライスを再水和。 </li><li> H＆E染色のために50％エタノール、4倍の脱イオン水、1×ヘマトキシリン​​、1×塩酸、電子を1倍、エタノールで2回、エタノールで3回、キシロールで70％96％4倍ガラス染色キュベットを準備thanol（13.7ミリリットル1 MのHCl広告200ミリリットルの50％エタノール）、1×タブ水、1×エオシン及び2×2プロパノール（100ミリリットルの脱イオン水中1グラムのエオシン）。 </li><li>場所はスライドを脱パラフィンし、再水和するキシロールIIにキシロールIおよび10分で10分をスライド。ディップは、エタノール50％エタノール70％、3回で3回、エタノールでエタノール96％I、3回で96％IIを3回スライドさせる。脱イオン水でスライドを回転させます。ヘマトキシリン​​染料を区別するために、塩酸 - エタノールで2倍を浸し。脱イオン水ですすいでください。ブルーイングのために、場所は、タブの水に5分をスライド。エオシン染色のために、場所はエオシンで1分をスライドし、脱イオン水で、その後洗い流してください。脱水、ディップスライドはエタノール96％エタノール中で70％、2×2×2-プロパノールII中で5分間、2プロパノールI中で5分間に置き、キシロール中で5分間IキシロールIIで5分。 H＆E染色した後、細胞核が赤く染色される青色、細胞質と細胞外マトリックスに染色される。 </li><li>抗原回復のために、キシレンでパラフィンを除去し、染色のためのスライスを再水和。スライスを配置スチームクッカーでと、予熱した10 mMのクエン酸緩衝液（pH 6）で20分間脱パラフィンし、再水和のスライスを調理。 </li><li>撥液性スライドマーカーペンまたはダイヤモンド針を有する洗浄緩衝液の代わりスライド（PBS緩衝液+ 0.5％ポリソルベート20）、円スライス。 </li><li>湿気チャンバー内でスライドを置きます。各スライスに100μlの3％過酸化水素溶液を添加することにより、内因性ペルオキシダーゼをブロックする。注意！皮膚や眼への接触の場合は非常に危険な。個人用保護具を取り扱ってください。洗浄は、洗浄緩衝液中でスライドする。 </li><li>一次抗体を適用し、室温で1時間インキュベートする。洗浄は、洗浄緩衝液で3回スライドさせる。ここから先、サンプルは暗所に保管する必要があります。 </li><li>特異的な抗原 - 抗体結合の検出のためにビオチン - ストレプトアビジンの検出システムを使用する。 </li><li> 1分間ヘマトキシリン​​で核を対比。スライドを脱水し、マウントします。 </li><li>逆顕微鏡を用いた画像のサンプル。 </li></ol></li></ol> &lt;Pクラス= &quot;jove_title&quot;&gt; 5。自動化された創傷装置を用いて損傷（AWD） <ol><li> 70％エタノールおよび紫外光で作業領域を消毒することでAWDを準備します。目的のサイズ（ 例えば、1ミリメートル）のドリルヘッドが装着されていることを確認します。 </li><li>滅菌ピンセットを用いて、オートクレーブ処理サンプルキャリアプレートの指定された領域に皮膚等価物を置きます。ドリルヘッド下のソケットにサンプルキャリアプレートを置きます。 </li><li>スピン速度：次のように創傷のパラメータを設定する制御ソフトウェアを使用して、15,000 rpmでの、浸透：1.5ミリメートル。推進：100ヘルツ。これらのパラメータは、個別に各サンプルタイプのために定義される必要がある。 AWDに創傷パラメータを転送します。 </li><li>手続きが負傷開始します。ソケットからサンプルキャリアプレートを取り外します。転送には、滅菌ピンセットを用いて培養に用いるインサートに戻すFTSEを負傷した。 </li><li>再生の調査のためのインキュベーター（37℃、5％のCO 2）で負傷FTSEの文化に進みます。アルternatively、任意の時点で、組織学的分析のためのモデルを使用しています。 </li></ol>

<ol>
	<li>Proksch, E., Brandner, J. M., Jensen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skin:+an+indispensable+barrier.">The skin: an indispensable barrier.</a> Exp Dermatol. 17, 1063-1072 (2008).</li><li>Guo, S., Dipietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+wound+healing.">Factors affecting wound healing.</a> J Dent Res. 89, 219-229 (2010).</li><li>Kirsner, R. S., Eaglstein, W. 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+wound+healing+process.">The wound healing process.</a> Dermatol Clin. 11, 629-640 (1993).</li><li>Cory, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratch-wound+assay.">Scratch-wound assay.</a> Methods Mol Biol. 769, 25-30 (2011).</li><li>Sun, T., Jackson, S., Haycock, J. W., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+skin+cells+in+3D+rather+than+2D+improves+their+ability+to+survive+exposure+to+cytotoxic+agents.">Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents.</a> J Biotechnol. 122 (3), 372-381 (2006).</li><li>Harrison, C. A., Heaton, M. J., Layton, C. M., Mac Neil, S. <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+an+in+vitro+model+of+tissue-engineered+human+skin+to+study+keratinocyte+attachment+and+migration+in+the+process+of+reepithelialization.">Use of an in vitro model of tissue-engineered human skin to study keratinocyte attachment and migration in the process of reepithelialization.</a> Wound Repair Regen. 14 (2), 203-209 (2006).</li><li>Wilkins, L. M., Watson, S. R., Prosky, S. J., Meunier, S. F., Parenteau, N. 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+bilayered+living+skin+construct+for+clinical+applications.">Development of a bilayered living skin construct for clinical applications.</a> Biotechnol Bioeng. 43 (8), 747-756 (1994).</li><li>Gangatirkar, P., Paquet-Fifield, S., Li, A., Rossi, R., Kaur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+3D+organotypic+cultures+using+human+neonatal+epidermal+cells.">Establishment of 3D organotypic cultures using human neonatal epidermal cells.</a> Nat Protoc. 2 (1), 178-186 (2007).</li><li>Boyce, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+substitutes+from+cultured+cells+and+collagen-GAG+polymers.">Skin substitutes from cultured cells and collagen-GAG polymers.</a> Med Biol Eng Comput. 36 (6), 791-800 (1998).</li><li>El-Ghalbzouri, A., Lamme, E. N., van Blitterswijk, C., Koopman, J., Ponec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+PEGT/PBT+as+a+dermal+scaffold+for+skin+tissue+engineering.">The use of PEGT/PBT as a dermal scaffold for skin tissue engineering.</a> Biomaterials. 25 (5), 2987-2996 (2004).</li><li>Maas-Szabowski, N., Shimotoyodome, A., 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=Keratinocyte+growth+regulation+in+fibroblast+cocultures+via+a+double+paracrine+mechanism.">Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism.</a> J Cell Sci. 112 (Pt 12), 1843-1853 (1999).</li><li>Coolen, N. A., Vlig, M., vanden Bogaerdt, A. J., Middelkoop, E., Ulrich, M. M. <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+an+in+vitro+burn+wound+model.">Development of an in vitro burn wound model.</a> Wound Repair Regen. 16 (4), 559-567 (2008).</li><li>Oberringer, M., Meins, C., Bubel, M., Pohlemann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro+wound+model+based+on+the+co-culture+of+human+dermal+microvascular+endothelial+cells+and+human+dermal+fibroblasts.">A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts.</a> Biol Cell. 99 (4), 197-207 (2007).</li><li>Cribbs, R. K., Luquette, M. H., Besner, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+model+of+partial+thickness+scald+burns+in+mice.">A standardized model of partial thickness scald burns in mice.</a> J Surg Res. 80 (1), 69-74 (1998).</li><li>Reed, J. T., Ghadially, R., Elias, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+type,+but+neither+race+nor+gender,+influence+epidermal+permeability+barrier+function.">Skin type, but neither race nor gender, influence epidermal permeability barrier function.</a> Arch Dermatol. 131 (10), 1134-1138 (1995).</li><li>Pilcher, B. 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=Keratinocyte+collagenase-1+expression+requires+an+epidermal+growth+factor+receptor+autocrine+mechanism.">Keratinocyte collagenase-1 expression requires an epidermal growth factor receptor autocrine mechanism.</a> J Biol Chem. 274 (15), 10372-10381 (1999).</li><li>Blank, I. H., Miller, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+separation+of+the+epidermis+from+the+dermis.">A method for the separation of the epidermis from the dermis.</a> J Invest Dermatol. 15 (1), 9-10 (1950).</li><li>El Ghalbzouri, 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=Fibroblasts+facilitate+re-epithelialization+in+wounded+human+skin+equivalents.">Fibroblasts facilitate re-epithelialization in wounded human skin equivalents.</a> Lab Invest. 84 (1), 102-112 (2004).</li><li>Vaughan, M. B., 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+reproducible+laser-wounded+skin+equivalent+model+to+study+the+effects+of+aging+in+vitro.">A reproducible laser-wounded skin equivalent model to study the effects of aging in vitro.</a> Rejuvenation Res. 7 (2), 99-110 (2004).</li><li>Geer, D. J., Swartz, D. D., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+model+of+wound+healing+based+on+transplanted+tissue-engineered+skin.">In vivo model of wound healing based on transplanted tissue-engineered skin.</a> Tissue Eng. 10 (7-8), 1006-1017 (2004).</li><li>Bell, 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=The+reconstitution+of+living+skin.">The reconstitution of living skin.</a> J Invest Dermatol. 81 (1 Suppl), 2s-10s (1983).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> Br J Dermatol. 111, 219-222 (1984).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat Rev Mol Cell Biol. 8 (10), 839-845 (2007).</li></ol>

The authors have nothing to disclose.

インビトロで細胞は通常、細胞がプラスチック表面に付着した二次元細胞培養物中で増殖させる。しかしながら、これらの培養条件は、細胞が生体内で成長する生理学的三次元の条件を反映していない。三次元の条件の下では、細胞は、天然の細胞 - 細胞および細胞 - マトリックスの添付ファイルを形成することができ、三次元に遊走する。特にin vivoでの状況の類似治?...

皮膚は体の最大の器官である。これは、外部環境と内部器官の間の障壁を作成します。さらに、皮膚は、流体損失、環境の影響、傷害および感染から身体を保護し、体温1を調節するのに役立つ。 、その暴露の場所に、皮膚は、多くの場合、機械的、熱的または化学的な外傷の影響を受けます。皮膚の自己修復が可能な一般的であるが、そのような感染症、酸素、および静脈充足などの複数の局所因子は、創傷治癒障害をもたらすことができる。創傷治癒はまた、肥満、アルコール依存症、喫煙、薬物療法、栄養、糖尿病などの疾患などの全身的な要因によって干渉することができます。2 （i）の炎症段階、（ii）の増殖および（iii）リモデリング段階：創傷治癒の過程は3段階に分けることができる。皮膚への損傷時には、複雑な信号カスケードは、傷の閉鎖につながる、開始します。3損傷後、創傷は出血し、血餅が形成される。線維芽細胞は、血液凝固に移動し、その後年間で改装された新しい組織と交換してください。 皮膚の修復の基礎となる生物学的プロセスの現在の理解は限られている。小動物およびブタモデルは創傷治癒を研究するために使用されてきた。しかし、これらの結果は、直接に種特異的な違いに起因するヒトに転送することができない。これらのin vivoモデルに加えて、創傷治癒のいくつかの側面は、不死化細胞株または初代細胞に基づくin vitroで単層培養物のスクラッチを介して、創傷の状況をシミュレートすることによって調べることができる4これらのスクラッチモデルは高度に標準化されているが、十分に反映していない複合体のin vivo生理学5の二次元モデルの他には、三次元のヒト皮膚同等物は、皮膚科学研究のために開発されている。これらのモデルの皮膚部分は、葛ある脱細胞化真皮、6コラーゲンハイドロゲル、7,8グリコサミノグリカン9または合成材料を含む様々な足場を使用してnerated。10これらの皮膚同等物、上皮間葉相互作用11の役割、再上皮、線維芽細胞およびケラチノサイトの間に細胞のクロストークや採用異なる成長因子の影響を研究することができる。さらに、これらのモデルは、線維芽細胞が創傷領域内に移動し、どのように走化性因子は、組織再生にどのように影響するかについての新しい知識を得るために有用である。12 創傷治癒モデル自体の唯一ではない世代は挑戦的であるだけでなく、モデル性の高い標準化された傷を確立することは問題がある。傷を作成するための一般的な技術は、スクラッチテスト、13火傷、14テープ摩耗、15熱傷、16吸引水疱、17液体窒素、18レーザー、19アール</SUP&gt;メス、18メッシャー6と生検パンチ。20これらのメソッドのほとんどは、同じ落とし穴を持っている。手動で実装けがを標準化し、複数のテストの間で再現することが困難である。サイズ、形状、および傷の深さは、研究の間で変化するので、研究データの品質を損なう。定義された皮膚の創傷のためのレーザーの使用は、比較的容易に標準化されたが、火傷を模倣する状況をもたらすことができる。レーザーによって加えられた熱は、壊死組織につながることができ、タンパク質の変性、血小板の凝集や血管収縮を引き起こす可能性があります。 別のアプローチでは、我々は、私たちは、無菌条件下で定義されており、正確な皮膚創傷を生成することができ、自動化創傷デバイス（AWD）を、開発しました。浸透深さと速度のようなパラメータを創傷ならびにドリルヘッドの回転を規制することができる。本研究では、家の中で開発された全層皮膚等価物とAWDを組み合わせた（FTGangatirkar らによって公開プロトコルに匹敵するSE）。8皮膚同等物の真皮層は、I型コラーゲンは、ヒドロゲルに埋め込 ​​まれたヒト皮膚線維芽細胞（HDF）、から構成されている。真皮層の上に、ヒト表皮ケラチノサイト（HEK）は播種。二週間以内に、気液界面でのHEKは、いくつかの重要な細胞層からなる表皮と角質層を構築する。このモデルの生成に加えて、本研究では、FTSEで定義され、正確な傷を作成するためのAWDの使用を示している。

<table><tbody><tr><td>Trypsin EDTA (1:250) 0.5% in DPBS</td><td>PAA</td><td>L11-003</td><td>0.05%</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Collagen (6 mg/ml in 0.1% acetic acid)</td><td>Produced in house</td><td></td><td></td></tr><tr><td>Fibronectin Human Protein, Plasma (50&amp;nbsp;&amp;micro;g/ml)</td><td>Life Technologies</td><td>33016-015</td><td></td></tr><tr><td>Inserts</td><td>Nunc</td><td>140627</td><td></td></tr><tr><td>6 well plate&amp;nbsp;</td><td>Nunc</td><td>140685</td><td></td></tr><tr><td>24 well plate</td><td>Nunc</td><td>142485</td><td></td></tr><tr><td>Microscope slides</td><td>R. Langenbrinck</td><td>03-0070</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Fibroblasts culturing (500 ml):&lt;/strong&gt;</td></tr><tr><td>DMEM, high glucose</td><td>Life Technologies</td><td>11965-092</td><td>89% (445 ml)</td></tr><tr><td>Fetal bovine serum</td><td>Bio &amp;amp; Sell</td><td>FCS.ADD.0500</td><td>10% (50 ml)</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>1% (5 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Keratinozyten culture medium (500 ml):&lt;/strong&gt;</td></tr><tr><td>Keratinocyte Growth Medium 2</td><td>Promocell&amp;nbsp;</td><td>C-20111</td><td>89% (445 ml)</td></tr><tr><td>Keratinocyte Growth Medium 2 SupplementPack</td><td>Promocell&amp;nbsp;</td><td>C-39011</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>1% (5 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Gel neutralization solution (250 ml):&lt;/strong&gt;</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium, high Glucose Powder with L-Glutamine</td><td>PAA</td><td>G0001,3010</td><td>93% (232.5 ml)</td></tr><tr><td>Chondroitin sulfate sodium salt from shark cartilage</td><td>Sigma</td><td>C4384-1g</td><td>1% (2.5 ml)</td></tr><tr><td>Fetal bovine serum</td><td>Bio &amp;amp; Sell</td><td>FCS.ADD.0500</td><td>3% (7.5 ml)</td></tr><tr><td>HEPES</td><td>Sigma&amp;nbsp;</td><td>H3375-1kg</td><td>3% (7.5 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Skin model submers medium (500&amp;nbsp;ml):&lt;/strong&gt;</td></tr><tr><td>Keratinocyte Growth Medium 2</td><td>Promocell&amp;nbsp;</td><td>C-20111</td><td></td></tr><tr><td>Keratinocyte Growth Medium 2 SupplementPack</td><td>Promocell&amp;nbsp;</td><td>C-39011</td><td></td></tr><tr><td>Fetal calf serum</td><td>Bio &amp;amp; Sell</td><td>FCS.ADD.0500</td><td>5%-2% (25 ml-10 ml)&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>1% (5 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Skin model air-liquid interface medium (500 ml):&lt;/strong&gt;</td></tr><tr><td>Keratinocyte Growth Medium 2</td><td></td><td></td><td></td></tr><tr><td>Keratinocyte Growth Medium 2 Supplement Pack</td><td>Promocell&amp;nbsp;</td><td>C-39011</td><td>adding only supplements: insulin, hydrocortisone, epinephrine, transferrin, CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>1% (5 ml)</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (300 mM)</td><td>Sigma&amp;nbsp;</td><td>C7902-500g</td><td>0.62% (3.1 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Histology:&lt;/strong&gt;</td></tr><tr><td>IHC-Kit DCS SuperVision 2 HRP</td><td>DCS</td><td>PD000KIT</td><td></td></tr><tr><td>Vimentin antibody</td><td>Abcam</td><td>ab92547</td><td></td></tr><tr><td>CK14 antibody</td><td>Sigma&amp;nbsp;</td><td>HPA023040-100&amp;micro;l</td><td></td></tr><tr><td>CK10 antibody</td><td>Dako</td><td>M7002</td><td></td></tr><tr><td>Filaggrin antibody</td><td>Abcam</td><td>ab81468</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;H&amp;amp;E staining:&lt;/strong&gt;</td></tr><tr><td>Mayer&amp;acute;s Haemalaun</td><td>AppliChem</td><td>A0884,2500</td><td></td></tr><tr><td>Xylol</td><td>Sigma Aldrich</td><td>296325-4X2L</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>32205-4X2.5L</td><td></td></tr><tr><td>HCl</td><td>Sigma Aldrich</td><td>H1758-500ML</td><td></td></tr><tr><td>Eosin</td><td>Sigma Aldrich</td><td>E4009-5G</td><td></td></tr><tr><td>2-Propanolol</td><td>Sigma Aldrich</td><td>I9516-500ML</td><td></td></tr><tr><td>Mounting Medium</td><td>Sigma Aldrich</td><td>M1289-10ML</td><td></td></tr></tbody></table>

generation of a three-dimensional full thickness skin equivalent and automated wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

孤立したHDFとHEKは、形態で、典型的なマーカーの発現の両方で異なっている。 HEKの形態は敷石状形態によって説明することができるのに対し、HDFは、典型的な紡錘状の形態を示した。細胞は、FTSEのためにそれらを使用する前に、免疫組織化学的染色（ 図1）によって特徴付けられた。 HDFは、線維芽細胞のマーカーであるビメンチン（ 図1A）に陽性である。主要HEKは?...

Watch this Scientific Journal Video about Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding at JoVE.com

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

generation-three-dimensional-full-thickness-skin-equivalent-automated

Research

JoVE Journal

Bioengineering

18.7K ビュー.  University Hospital Würzburg. The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility. 

ビデオ: Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

失活ヒト真皮を用いて遺伝子組換え器官皮膚培養物の生成

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

発生生物学

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

生体材料の血管新生を評価するために、完全な皮膚欠損モデル<em&gt;インビボ</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

生物工学

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

定量的、皮膚が創傷管理のためのコンフォーマルスキンのような電子システムの作製と評価

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

2Dと3Dの肌の改善<em&gt;インビトロ</em分子クラウディングの使用&gt;モデル

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

皮膚修復を正確に評価するヒトEx vivo創傷モデルと全山染色法

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

3次元の再構築されたヒト表皮を大規模に育成

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

自己組み立て型血管化ヒト皮膚等価物の生成

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

マイクロマシンマイクロ流体プラットフォームにおける簡単な3次元スキンオンチップモデルの生成

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

<meta charset="utf8"></head><body>効率的な3Dスキンモデルによる柔軟な研究アプリケーション

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

<meta charset="utf8"></head><body>著者スポットライト:費用対効果の高い3D細胞モデルによるスキンモデルの多様性の強化

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

2次元(2D)単一培養から3次元(3D)多文化まで、さまざまな用途に対応するスキンモデルの構築

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

三次元の全層皮膚等価物と自動傷つけ処理の発生

要約

さらに動画を探す

この動画の章

失活ヒト真皮を用いて遺伝子組換え器官皮膚培養物の生成

生体材料の血管新生を評価するために、完全な皮膚欠損モデル

定量的、皮膚が創傷管理のためのコンフォーマルスキンのような電子システムの作製と評価

2Dと3Dの肌の改善

皮膚修復を正確に評価するヒトEx vivo創傷モデルと全山染色法

3次元の再構築されたヒト表皮を大規模に育成

自己組み立て型血管化ヒト皮膚等価物の生成

マイクロマシンマイクロ流体プラットフォームにおける簡単な3次元スキンオンチップモデルの生成

2次元(2D)単一培養から3次元(3D)多文化まで、さまざまな用途に対応するスキンモデルの構築

2次元(2D)単一培養から3次元(3D)多文化まで、さまざまな用途に対応するスキンモデルの構築