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1。の単離脂肪由来幹細胞（ASC） 注：特に断りのない限り、すべての手順は室温で行った。 <ol><li>ラット腎周囲および副睾丸脂肪を分離し、前述の6、1％ウシ胎児血清（FBS）を含有する滅菌ハンクス緩衝塩溶液（HBSS）で洗浄する。 </li><li>組織をミンチし、室温で8分間500グラムで50 mLのチューブと遠心分離機に1％FBSを含むHBSS 25 mLに1〜2グラムを転送します。 </li><li>浮遊脂肪組織の層と125 mLの三角フラスコへの転送を収集し、37℃で45分間HBSSでコラゲナーゼII型（200 U / mL）を25 mLで扱う°オービタルシェーカー（125 rpm）でのC。 </li><li>慎重に液体画分（油と脂肪層の下）を削除し、100μmと70μmのナイロンメッシュフィルターを順次フィルタリングします。室温で10分間500gで濾液を遠心し、supernatanを吸引tと25 mLのHBSSでペレットを2回洗浄する。 </li><li> MesenPRO RS成長サプリメントを添加した増殖培地50 mLの細胞ペレット（MesenPRO RS基礎培地）、抗生物質抗真菌剤（ペニシリンG、100μg/ mLの硫酸ストレプトマイシン、および0.25μg/ mLのアムホテリシンBの100 U / mL）を懸濁し、2 mM L-グルタミンとピペットセル2 T75フラスコ（25 / mLのフラスコ）に変換します。 </li><li>文化37°C（ASCは、すべての実験に使用されているパッセージ2-4）で5％CO 2の加湿インキュベーター中でASC。 </li></ol> 2。キトサンマイクロスフェア（CSM）を準備 注：特に断りのない限り、すべての手順は室温で行った。 <ol><li> CSMは、当社の以前のプロトコル6を使用してイオンコアセルベーション技術とともに油中水型乳化プロセスによって調製される。大豆から成る油相混合液100 mLにキトサンの水溶液（0.5 M酢酸の3％（w / v）のキトサンの6 mL）を乳化油、n-オクタノール（1:2 v / v）で、5％ソルビタンモノオレエート（スパン80）、乳化剤、反対方向に、オーバーヘッド（1700 rpm）と同時に磁気攪拌（1000回転）を使用します。初期の架橋が発生する前に上に形成されたミセル溶液中に残っていると底に沈殿することはできませんミ​​キシングを保証し、この二重の方法。さらに、デ集約ミセル形成とrigidization中にキトサンをで磁気攪拌棒を支援します。 </li><li>混合物を連続的に安定した油中水型エマルションが得られるまで約1時間撹拌する。イオン架橋は4時間（24 mLの合計）のn-オクタノール15分毎に1％（w / v）の水酸化カリウムを1.5 mLの添加で開始されます。 </li><li>架橋反応の完了後、徐々に混合物を含むCSMの油相をデカントし、直ちにアセトン100mLに球を追加します。油相が完全に除去されるまで、アセトンで球を洗浄します。 </li><li>真空デシケーターと肛門に回復した球体を乾燥させさらなる処理なしyze。平均CSMの粒径、ミリグラム当たりの表面積と単位体積は、粒子サイズアナライザーを用いて決定した。 </li><li>その後の実験では、残留塩を除去し、無水アルコールで滅菌する滅菌水でCSM 3回洗浄します。 </li></ol> 3。 CSMの遊離アミノ基の数を決定する 注：特に断りのない限り、すべての手順は室温で行った。 <ol><li> BubinsとOfner 7トリニトロベンゼン（TNBS）酸アッセイを用いてイオン架橋後のCSMに存在する遊離アミノ基の数を決定します。 40°Cで4時間、50 mLのガラス管に0.5％TNBS溶液1mLをマイクロスフェアの5mgをインキュベートすると2h、60℃で6N塩酸3 mLを加えて加水分解する。 </li><li>室温にサンプルを冷却し、脱イオン水5mLを追加することによって、自由にTNBSを抽出し、酢酸エチル10 mLのエーテル。 </li><li> ℃で15分間水浴中で室温まで冷却し、残留エーテルを蒸発させ、水15 mLで希釈して40に水相5 mLを温める。 </li><li>ブランクとキトサンのアミノ基の合計数を決定するためにCSMの準備のために使用されるキトサンなく、TNBS溶液を用いて、分光光度計で345 nmの吸光度を測定します。キトサンへの相対的なCSMの遊離アミノ基の数を推定する。 </li></ol> 4。 CSMの読み込みASC 注：特に断りのない限り、すべての手順は室温で行った。 <ol><li>一晩滅菌HBSSでセクション2.5から滅菌CSMの5mgを平衡化し、8μmの細孔径の膜培養プレートインサート（24ウェルプレート）に追加します。 </li><li> CSMはメンブレンに定住した後、慎重にHBSSを吸引し、目に増殖培地の挿入、700μLの内部に増殖培地300μLを追加インサートの外側の電子。 </li><li>培養プレートのインサート内部CSM上の成長培地と種子の200μLの適切な濃度で再懸濁し、ASC（1×10 4から4×10 4）。カルチャーインサート内の培地の最終量は、播種後、500μLです。 </li><li> 37℃5％CO 2の加湿インキュベーター内で24時間CSM上でASCシードをインキュベート℃に</li></ol> 5。 CSMでのASCの読み込みと細胞生存率の割合を決定する 注：特に断りのない限り、すべての手順は室温で行った。 <ol><li>インキュベーション後、インサート膜に移行した細胞を乱すことなく滅菌1.5 mlのマイクロ遠心チューブにASCにロードされたCSMを収集します。 </li><li>残留培地を除去し、チューブに新鮮な増殖培地250μLを追加します。 </li><li>各チューブに[3 - （4,5 - dimethylthiozole -2 - イル）-2,5 - ジフェニルテトラゾリウムブロマイド]を25μLMTTを追加する溶液（5 mg / ml）と37℃5％CO 2の加湿インキュベーター中で4時間インキュベート℃、 </li><li>インキュベーション後、培地を除去し、ジメチルスルホキシドの250μLを追加し、ホルマザンの複合体を可溶化するために2から5分間ボルテックス混合物を。 </li><li> 5分間2700×gでCSMを遠心し、その基準として630 nmを用いて570 nmで測定した上清の吸光度を決定します。 </li><li>実行可​​能なASCの知られている番号から得られた吸光度値からの相対CSMに関連付けられている細胞数を決定します。 </li></ol> 6。 ASC-CSM-組み込みコラーゲンゲルを特徴付ける 注：特に断りのない限り、すべての手順は室温で行った。 <ol><li> 2 N NaOHを用いて6.8にpHを調整した後、Bornstein 8とフィブリル化の方法に従って、ラットの尾の腱から抽出された1型コラーゲン（7.5 mg / ml）と混合ASCにロードされたCSM（5 mgを≈2×10 4細胞を含む）。 </lI&gt; <li> 12ウェルプレートにフィブリル化コラーゲン-ASC-CSM混合物を追加し、37℃5％CO 2の加湿インキュベーター中で30分間インキュベート℃、 </li><li>完全に心房細動の後、37℃で最大5％CO 2の加湿インキュベーター内で14日間のためにコラーゲン-ASC-CSMゲルをインキュベート</li><li>ゲルにCSMからの細胞の放出と移行は、標準的な顕微鏡技術を用いて観察した。 </li></ol> 7。代表的な結果本研究では、コラーゲンゲル足場にキトサンミクロスフェア（CSM）から幹細胞を提供するためにin vitroでの戦略を開発しました。均一な大きさ（直径175から225μm）と組成の多孔CSMは、細胞のキャリア（ 図2）として調製し使用しました。 CSMとASCをインキュベートすると、細胞は、CSMの2×10 4 cells/5mgの濃度で接続されている。糸状仮足を延長しながら細胞は、マイクロスフェアを介して拡散することができましたミクロスフェア（ 図3）の多孔質の隙間に。セルにロードされたCSMは、コラーゲンゲルと混合した後、細胞はすぐにゲル（ 図4）に移行し始めました。 <img alt="表1に" src="/files/ftp_upload/3624/3624table1.jpg" /> 表1。幹細胞デリバリーシステムのキトサンとコラーゲンの使用のための生物学的利点があります。 <img alt="図1" src="/files/ftp_upload/3624/3624fig1.jpg" /> 図1回路図は、ASC-CSMロードされたコラーゲンの足場の二重使用のための全体的な戦略を描いた。 図2、3、4、画像の解釈を支援するための回路図内で注釈が付けられています。 <img alt="図2" src="/files/ftp_upload/3624/3624fig2.jpg" /> 図2の模式図では、キトサンミクロスフェアに幹細胞を播種の過程を描いた。 PROCμmの孔径の膜培養プレートインサート - ESSは8 CSMと共培養ASCが含まれます。 24時間後、微小球は、挿入から削除され、生体マトリックス中に埋め込むための準備が整いました。 <img alt="図3" src="/files/ftp_upload/3624/3624fig3.jpg" /> 図3。ASCとCSM-ロードされた形態学的特性。パネルBは共焦点蛍光顕微鏡によって得られた画像を重ね合わせたビューの同じフィールドを示しているパネルは、ASCにロードされたのCSMの光顕微鏡写真を示しています。 ASCは、カルセインAM（緑色）がプリロードされた。パネルDは、ミクロスフェア（アスタリスク）にロードされた細胞を示しているパネルCは、無負荷時のマイクロスフェアのSEM像のイメージを示しています。パネルEのTEMイメージがアンロードされたミクロスフェアの断面を示しています。毛穴や割れ目の多数は、フェア全体に配置されています。パネルFは、細胞とのクロス切片ミクロスフェア（矢印）を接続し、CREに糸状仮足を延長を示しています。悪徳。オリジナル倍率：A＆B = 70X、C = 500分の1、D = 2,000×、E＆F = 2,500 X。 <img alt="図4" src="/files/ftp_upload/3624/3624fig4.jpg" /> 図4三次元コラーゲンスキャフォールドにCSMからASCの移行。パネルAおよびBは、細胞が離れてミクロスフェアからの移行で、3日目にコラーゲンマトリックス（矢印）にCSMを描いています。パネルBは、12日後に同様の文化を示しています。透過型電子顕微鏡（TEM）画像は、ミクロスフェア（矢印）からの移行細胞とのクロス切片であったミクロスフェアを表示CとDにC、D、およびEアスタリスクで示されている。パネルDの高倍率は、パネルEに示すように、コラーゲン線維（挿入図）に接続されているセルの糸状仮足を示しています。オリジナル倍率：A＆B = 100倍、C＆D = 6,000 X; E = 20,000、はめ込み= 150,000 X。 <img alt="図5" src="/files/ftp_upload/3624/3624fig5.jpg" /> 図5。回路図は、再生医療とドラッグデリバリーにおけるCSMの広大な使用方法を描いた。

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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFATS+collection:+Human+adipose-derived+stem+cells+seeded+on+a+silk+fibroin-chitosan+scaffold+enhance+wound+repair+in+a+murine+soft+tissue+injury+model.">IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model.</a> Stem Cells. 27, 250-258 (2009).</li><li>Machado, C. B., Ventura, J. M., Lemos, A. F., Ferreira, J. M., Leite, M. F., Goes, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+chitosan-gelatin-chondroitin+porous+scaffold+improves+osteogenic+differentiation+of+mesenchymal+stem+cells.">3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells.</a> Biomed. Mater. 2, 124-131 (2007).</li></ol>

なし競合する経済的利益は存在しません。 免責事項ここに記載されている意見やアサーションは、作者の私的見解であり、公式、または国防省、または米国政府の部門の意見を反映して解釈されるべきではありません。著者らは、米国政府の従業員であり、この作品は、その職務の一部として作成されました。すべての作業は、米陸軍医学研究および資材コマンドでサポートされていました。本研究では、米陸軍医学研究および資材コマンド機関審査委員会によって審査、承認されたプロトコルの下で実施され、承認されたプロトコルに準拠していた。

幹細胞ベースの治療の主要なハードルは、修理のために指定された領域への細胞の送達のための効率的な方法を開発しています。患者変動への患者の、組織の種類、損傷の大きさと深さのために、幹細胞を提供する方法論は、ケースバイケースで決定する必要があります。マトリックス内の幹細胞を埋め込み、創傷部位にそれらを提供する組織工学のための次の論理的なアプローチであるよ?...

<table border="1" ><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ハンクスBalancedSalt溶液（HBSS） </td><td>ギブコ</td><td> 14175 </td><td>消耗品</td></tr><tr><td>ウシ胎児血清</td><td>ハイクローン</td><td> SH30071.03 </td><td>消耗品</td></tr><tr><td>コラゲナーゼタイプII </td><td> Sigma-Aldrich社</td><td> C6685 </td><td>消耗品</td></tr><tr><td> 70μmのナイロンメッシュフィルター</td><td> BDバイオサイエンス</td><td> 352350 </td><td>消耗品</td></tr><tr><td> 100μmのナイロンメッシュフィルター</td><td> BDバイオサイエンス</td><td> 352360 </td><td>消耗品</td></tr><tr><td> MesenPRO増殖培地システム</td><td>インビトロジェン</td><td> 12746-012 </td><td>消耗品</td></tr><tr><td> L-グルタミン酸</td><td>ギブコ</td><td> 25030 </td><td>消耗品</td></tr><tr><td> T75組織培養フラスコ</td><td> BDバイオサイエンス</td><td> 137787 </td><td>消耗品</td></tr><tr><td>キトサン</td><td> Sigma-Aldrich社</td><td> 448869 </td><td>消耗品</td></tr><tr><td>酢酸</td><td> Sigma-Aldrich社</td><td> 320099 </td><td>消耗品</td></tr><tr><td> n-オクタノール</td><td>アクロスオーガニック</td><td> 150630025 </td><td>消耗品</td></tr><tr><td>ソルビタン、モノオレイン酸</td><td> Sigma-Aldrich社</td><td> S6760 </td><td>消耗品</td></tr><tr><td>水酸化カリウム</td><td> Sigma-Aldrich社</td><td> P1767 </td><td>消耗品</td></tr><tr><td>アセトン</td><td>フィッシャー·サイエンティフィック</td><td> L-4859 </td><td>消耗品</td></tr><tr><td>エタノール</td><td> Sigma-Aldrich社</td><td> 270741 </td><td>消耗品</td></tr><tr><td>トリニトロベンゼンスルホン酸</td><td> Sigma-Aldrich社</td><td> P2297 </td><td>消耗品</td></tr><tr><td>塩酸</td><td> Sigma-Aldrich社</td><td> 320331 </td><td>消耗品</td></tr><tr><td>エチルエーテル</td><td> Sigma-Aldrich社</td><td> 472から484 </td><td>消耗品</td></tr><tr><td> 8μmの組織培養プレートインサート</td><td> BDバイオサイエンス</td><td> 353097 </td><td>消耗品</td></tr><tr><td> 1.5 mlのマイクロ遠心チューブ</td><td>フィッシャー</td><td> 05-408-129 </td><td>消耗品</td></tr><tr><td> MTT試薬</td><td>インビトロジェン</td><td> M6494 </td><td>消耗品</td></tr><tr><td>ジメチルスルホキシド</td><td> Sigma-Aldrich社</td><td> D8779 </td><td>消耗品</td></tr><tr><td> Qtracker細胞標識キット（Qトラッカー655） </td><td>分子プローブ</td><td> Q2502PMP </td><td>消耗品</td></tr><tr><td> 1型コラーゲン</td><td> Travigen </td><td> 3447-020-01 </td><td>消耗品</td></tr><tr><td>水酸化ナトリウム</td><td> Sigma-Aldrich社</td><td> S8045 </td><td>消耗品</td></tr><tr><td> 12ウェル組織培養プレート</td><td> BDバイオサイエンス</td><td> 353043 </td><td>消耗品</td></tr><tr><td>遠心分離</td><td>エッペンドルフ</td><td> 5417R </td><td>機器</td></tr><tr><td>オービタルシェーカー</td><td>ニューブランズウィック州Scienctific </td><td> C24 </td><td>機器</tD&gt; </tr><tr><td>空気-5％CO 2の加湿インキュベータ</td><td>サーモフィッシャーサイエンティフィック</td><td> 370 </td><td>機器</td></tr><tr><td>オーバーヘッドスターラー</td><td> IKA </td><td> Visc6000 </td><td>機器</td></tr><tr><td>マグネチックスターラー</td><td>コー​​ニング</td><td> PC-210 </td><td>機器</td></tr><tr><td>減圧デシケーター</td><td> - </td><td> - </td><td>機器</td></tr><tr><td>粒径分析器</td><td>マルバーン</td><td> STP2000 Spraytec </td><td>機器</td></tr><tr><td>水浴</td><td>フィッシャー·サイエンティフィック</td><td> Isotemp210 </td><td>機器</TD&gt; </tr><tr><td>分光光度計</td><td>ベックマン</td><td>ベックマン·コールターDU800UV/Visible分光光度計</td><td>機器</td></tr><tr><td>渦</td><td> Diagger </td><td> 3030a </td><td>機器</td></tr><tr><td>マイクロプレートリーダー</td><td>モレキュラーデバイス</td><td> SpectraMax M2 </td><td>機器</td></tr><tr><td>光/蛍光顕微鏡</td><td>オリンポス</td><td> IX71 </td><td>機器</td></tr><tr><td>共焦点顕微鏡</td><td>オリンポス</td><td> FV-500レーザー走査型共焦点顕微鏡</td><td>機器</td></tr><tr><td>走査型電子顕微鏡</td><td>カールツァイスマイクroImaging </td><td>レオ435 VP </td><td>機器</td></tr><tr><td>透過型電子顕微鏡</td><td> JEOL </td><td> JEOL 1230 </td><td>機器</td></tr></tbody></table>

constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

多能性幹細胞は再生医療1-3の分野で非常に有用であることが示されている。しかし、組織再生に効果的にこれらの細胞を使用するためには、変数の数を考慮する必要があります。これらの変数が含まれます：総体積と移植部位の表面積、組織の機械的性質および血管新生の量と細胞外マトリックスのコンポーネントが含まれています。組織の微小環境を、。宿主組織を模倣した機械的強度を維持しながら、したがって、これらの細胞を提供するために使用される材料は、定義された化学組成を持つ生体適合性でなければなりません。これらの材料はまた、酸素と栄養細胞が付着し、増殖するための有利な微小環境を提供するために透過性でなければなりません。キトサン、優れた生体適合性を有するカチオン性多糖類は、容易に化学的に修飾されたおよび in vivo Mac でと結合する高親和性を持つことができますromolecules 4-5。キトサンは、細胞接着、遊走および増殖のための基質として機能できるように、細胞外マトリックスのグリコサミノグリカン部分を模倣しています。本研究では、三次元足場6ベースのコラーゲンに脂肪由来幹細胞（ASC）を提供するミクロスフェアの形でキトサンを利用しています。理想的な細胞へのミクロス比はインキュベーション時間とロードすることができましたセルの最大数を達成するために、細胞密度を基準にして決定した。 ASCは、キトサンマイクロスフェア（CSM）に播種されると、それらはコラーゲン足場に埋め込まれており、長期間の培養で維持することができます。要約すると、この研究では、正確に三次元生体材料足場内で幹細胞を提供するメソッドを提供します。

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

現在の幹細胞治療の主要なハードルは、ホスト組織にこれらの細胞を提供するための最も効果的な方法を決定しています。ここでは、その多能性を維持するために、脂肪由来幹細胞を可能にしながら、アプローチの効率的かつ単純であるキトサンベースの配信方法について説明します。

幹細胞にロードされたキトサンミクロスフェアの配信のためにコラーゲンハイドロゲルの構築

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

constructing-collagen-hydrogel-for-delivery-stem-cell-loaded-chitosan

Research

JoVE Journal

Bioengineering

16.7K ビュー.  United States Army Institute of Surgical Research. 現在の幹細胞治療の主要なハードルは、ホスト組織にこれらの細胞を提供するための最も効果的な方法を決定しています。ここでは、その多能性を維持するために、脂肪由来幹細胞を可能にしながら、アプローチの効率的かつ単純であるキトサンベースの配信方法について説明します。

ビデオ: Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

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

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

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 ...

生物工学

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. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

変性コラーゲンストランドを画像化する<em&gt;インビボ</em&gt;と<em&gt;生体外</em&gt;リテーナ入りコラーゲン模倣ペプチド写真·トリガブリダイゼーション経由

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

用アメロゲニン - キトサンハイドロゲルの開発<em&gt;体外</em高密度インターフェイス付き&gt;エナメル再成長

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

マイクロスケール渦アシストシーケンシャル分子送達用エレクトロポ

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

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 ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

腎臓皮質細胞外マトリックス由来ハイドロゲルを製造

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an injury leading to delayed wound healing. We selected doxycycline (DOX), as a potential drug of choice, due to its anti-bacterial properties along with its reported anti-inflammatory properties. The current study aims to formulate DOX loaded collagen-chitosan non-crosslinked (NCL) &amp; crosslinked (CL) scaffolds and evaluate their healing ability in diabetic conditions. The characterization result of scaffolds reveals that the DOX-CL scaffold holds ideal porosity, a low swelling &amp; degradation rate, and a sustained release of DOX compared to the DOX-NCL scaffold. The in vitro studies reveal that the DOX-CL scaffold was biocompatible and enhanced cell growth compared with CL scaffold treated and control groups. The anti-bacterial studies have shown that the DOX-CL scaffold was more effective than the CL scaffold against the most common bacteria found in DW. Using the streptozotocin and high-fat diet-induced DW model, a significantly (p&#8804;0.05) faster rate of wound contraction in the DOX-CL scaffold treated group was observed compared to those in CL scaffold treated and control groups. The use of the DOX-CL scaffold can prove to be a promising approach for local treatment for DWs.

The prepared DOX-CL scaffold satisfied the prerequisites of an ideal DW dressing in mechanical strength, porosity, water absorption, degradation rate, sustained release, anti-bacterial, biocompatibility, and anti-inflammatory properties, which are considered to be essential for the recovery of damaged tissue in DWs.

糖尿病性創傷の加速治癒のためのドキシサイクリン装填されたコラーゲン-キトサン複合足場

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

末梢神経工学のためのグラフェンベースの3Dバイオハイブリッドヒドロゲルバイオインクの調製と特性評価

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

組織工学用途のためのサイズ制御およびエマルジョンフリーのキトサン-ジェニピンミクロゲルの製造

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...