著者らは、サザンイリノイ大学医学部から手術研究助成金のスタートアップ資金や部署を承認したいと思います。メモリアル医療財団補助金。

1。手術検索注：IRB承認がACL再建手術中にACL断端を収集するために得た。使用するACL断端は一般外科廃棄物として廃棄されたように、IRBの免除が与えられた。 20人の患者は、ACL断端を収集するために使用された。 <ol><li>患者への金融機関のプロトコールに従って、全身麻酔や手術前の抗生物質を管理します。 </li><li>止血帯を適用し、収縮期血圧が上100ミリグラムを膨らませる。 </li><li>仰臥位で患者を置く。 5ミリメートルポータルの30〜45°の屈曲で膝水平前外側切開を行い、30℃で膝蓋上パウチに関節鏡トロカールとカメラを挿入します。 </li><li>日常的な診断関節鏡検査を実行します。 </li><li>ノッチを入力して、引き裂かれたACLより膜性滑膜を創面切除。 </li><li> ACL断を識別するために、4.5ミリメートルのシェーバーとアブレータを使用してください。 </li><li>複数のSPEを収集するためにまっすぐに苦いダックビルを使用残りのACL断からcimens。 </li><li>滅菌容器に生理食塩水のサンプルを維持する。 </li><li>デコードや実験施設への最終的な配信のために病理学にサンプルを送る。 </li><li>患者を回復し、金融機関のプロトコールに従って術後ケアを行う。 </li></ol> 2。組織消化とHACLの分離<ol><li>人間のACL組織は、（リン酸緩衝生理食塩水）滅菌生理食塩水/ PBSでシャーレに病理から受信した転送。 </li><li> 1〜2ミリメートル3個に、滅菌ハサミで組織をミンチし、少なくとも3回の生理食塩水でみじん切り組織を洗う。 </li><li> DMEM/F-12中の0.4％コラゲナーゼで37℃で4-6時間、10％ウシ胎児血清（FBS）および1％ペニシリン/ストレプトマイシンを補充した（50/50、1×）培地で細分化した組織を消化</li><li> 5分間の千×gで細胞を遠心分離した後、培地中で細胞を再懸濁。 </li><li> Sそれから2〜3回培地で細胞を洗浄し、EED / 2〜3日のT-25フラスコ内の培養。 </li></ol> 3。細胞拡大、凍結融解<ol><li> T-25フラスコ内で2〜3日間培養した細胞を可視化するために光学顕微鏡を使用しています。 </li><li> 10％ウシ胎児血清（FBS）および1％ペニシリン/ストレプトマイシン（P / S）を補充したDMEM/F-12（50/50、1×）培地中37℃で細胞を維持する。 注意：細胞が接着および標準形態を示すときに、メディアを変更します。細胞を観察するために光学顕微鏡を使用しています。 </li><li> 7日目（〜1.3×10 6細胞/フラスコ）にPBSでコンフルエントの細胞を洗浄します。トリプシン（0.5グラム/ L）を添加し、5分間37℃で細胞をインキュベートする。トリプシンを中和するために懸濁された細胞にメディアを追加。 3、T-25フラスコの間のメディア細胞の混合物を分割します。 </li><li> PBSでコンフルエントの細胞を洗浄し、トリプシン（0.5グラム/ L）を追加し、DMSO、FBSおよび完全培地（DMEM/F-12培地+、10％FBS +1％P / S）を含む凍結培地で再懸濁します比率午前1時02分07秒。 </li><li>店細胞は、より長い時間保存される場合に細胞はヶ月以内に使用され、液体窒素中で説明する場合は-80℃での低温バイアル。 </li><li>再利用のために37℃の水浴中でバイアルを解凍する。 注：通常90から95パーセントの細胞が生存。 </li></ol> 4。2-Dポリ乳酸 - コ - グリコール酸（PLAGA）組織工学的足場の作製<ol><li> 20ミリリットルのシンチレーションバイアル中の12ミリリットルのジクロロメタンにPLAGAの1グラムを溶解し、一定速度（800 rpm）で8時間のためのソリューションをボルテックスする。 注意：グプタら詳細な説明15。 </li><li>フッ素系保護紙を敷いたガラスシャーレに溶解した溶液を転送します。 </li><li> 30分間真空フードの下にペトリ皿を置きます。室温で一晩、その後-20℃でペトリ皿のままにしておきます。ポリマーの薄膜を得るために、溶媒を完全に蒸発を確実にするために凍結乾燥機中でペトリ皿を置きます。 </li><li>ポリマーFIを配置します24時間デシケーター中LMS。 </li><li>直径12mmの円形ディスクにポリマーフィルムをカットし、必要になるまでデシケーターに保管してください。 </li></ol> 5。走査型電子顕微鏡（SEM） <ol><li>細胞（50,000細胞/ディスク）を洗って播種7日以降のPBSで制御TCPSとPLAGA足場に播種。 </li><li>細胞を固定し、後固定のための0.1M酸緩衝液中で2.5％のOsO 4を0.1M酸緩衝液中で1.5％のグルタルアルデヒドを使用してください。 </li><li> 0.1Mの酸緩衝液で固定した細胞を洗浄します。それぞれ15分、そして一晩ヘキサメチルジシラザン（HMDS）中でさらに乾燥シリアルエタノール脱水（50％、70％、80％、90％及び100％）を用いて固定した細胞を乾燥させる。 </li><li>ボタンバルブを、SEMスパッタコーティングシステムのチャンバーをベントし、天板を上げる。 </li><li>室内で乾燥させた試料を配置します。天板を下げます。 </li><li>チャンバを排気開始するために圧送するコントロールノブを切り替えます。 </li><li>オープンアルゴンリーク弁。待つ真空は0.05ミリバールまで低下させるには。 </li><li>金/パラジウムの薄い層（0.13ナノメートル）でコーティング乾燥した試料。 </li><li>その閉じた位置にアルゴンリーク弁を返します。 </li><li>オフにコントロールノブを切り替えます。 </li><li>ボタンバルブとチャンバーベント天板を上げる。 </li><li>サンプルを取り出して、24時間デシケーターに保管してください。 </li><li>走査型電子顕微鏡でサンプルを観察します。 </li></ol> 6。免疫蛍光染色<ol><li>細胞（50,000細胞/ディスク）を洗って播種7日以降のPBSで制御TCPSとPLAGA足場に播種。 </li><li> 10分間、70％エタノール（コールド）を用いて細胞を固定します。 </li><li> PBSで20分間、0.05％トリトンX-100を有する中の1％ウシ血清アルブミン（BSA）を用いて室温で固定した細胞をインキュベートする。 </li><li> 20分間室温で1％Tween中のサンプルを浸す。 </li><li>モノクローナルマウス抗β-アクチン抗体（1:400）を追加し、overnigh細胞をインキュベート4℃でのT </li><li> 0.05％のTweenで細胞を洗浄。細胞への二次抗体（抗マウスF（ab &#39;ヤギ）蛍光プローブ、1:400と結合したIgGの2断片）を添加し、室温で1時間インキュベートする。 </li><li> PBSで細胞を洗浄。核染色で細胞を染色し、80％グリセロールを使用してマウントします。 </li><li>共焦点顕微鏡で細胞を観察します。 </li></ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+multipotent+stem+cells+from+human+cruciate+ligaments.">Isolation and characterization of multipotent stem cells from human cruciate ligaments.</a> Cell Prolif. 42 (4), 448-460 (2009).</li><li>Matsumoto, T., 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=Isolation+and+characterization+of+human+anterior+cruciate+ligament-derived+vascular+stem+cells.">Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells.</a> Stem Cells Dev. 21 (6), 859-872 (2012).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

このhACL/2D足場研究の主な目的は、部分的なACLの涙の主要な修理を強化するために、パッチで得られた細胞を使用することでした。部分的なACLの涙の非手術管理が固定化、ブレーシング、進歩的リハビリテーションプログラム、および定期的なフォローアップの評価13,16,17の短期間を含むことができる。しかし、多くの研究がスポーツ選手での保存的治療が不十分な結果、失敗と関​?...

前十字靱帯（ACL）は膝の一般負傷した関節内靭帯である。約20万（ACL）の傷害は、米国で毎年報告されています。整形外科再建手術1,2,3,4のACL損傷OPTを経験した患者の75％以上。外科的介入は、しばしば本質的に治癒不良の可能性3に示されている。 ACL再建は、典型的には、自家移植片又は同種移植腱によって達成される。自家移植と同種移植片は、高い成功率を誇るように復興のためのゴールドスタンダードを表し、一次縫合の修理、他の治療オプションは、最大で94％で5,6,7の故障率を示している。 ACLの部分的な涙はすべてのACLの涙8の28％、10％に相当する。プロスペクティブ研究では、ノイスら 、よりACLの半分以上の部分に影響を与える涙を有する患者の50％が後、非ACL不全を完了するために進行していると推定手術治療9。他の研究では、持続的な不安定性を報告し、その損傷前の活動レベル9,10,11,12,13に戻ることができた患者の30％未満で機能の低下。治療の選択肢は限られており、保守的な様式、残りのACLの熱収縮、またはACL再建が含まれています。最近では、拡張現実、一次修理に対する関心が高まっています。これらの技術は、一次縫合糸修復14を強化するために生物学的製剤を使用しています。最近の研究では、間葉系様HACLを収穫しようとした31,32、移植の制限を回避するために幹細胞を誘導されたが、これらの細胞の妥当性と有効性はまだ不明です。組織工学用途のための理想的な細胞源は、非mesechymal HACLは、線維芽細胞に由来すると思われる。 現在の研究は、適切なマトリックス材料および操作された足場のための細胞源の同定に焦点を当てている。引き裂かれたACLは、suとして廃棄されることが標準的な手順である再建手術中rgical廃棄物は、しかし、この損傷を受けた靭帯は、ACL置換を設計し、理想的な組織を開発し、強化するために必要な細胞の取得質源であってもよい。私たちの研究室では、これらを採取HACL由来細胞のin vitroでの拡大のためのプロトコルを開発しました。 HACL由来細胞を注入された設計の2Dマトリックスを使用して、我々は潜在的に部分的なACLの修復を増強し、引き裂かれた靭帯を強化する可能性がパッチを設計しました。

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			<td height="21" style="height:21px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:507px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri-dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">08-757-103C</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP3994</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Collagenase</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">17018-029</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">DMEM/F-12</td>
			<td style="width:139px;">Cellgro</td>
			<td style="width:143px;">10-092-CV</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">10082</td>
			<td>Store at -800C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-602E</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Tubes- 15 ml</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T-25 flasks</td>
			<td style="width:139px;">BD Falcon</td>
			<td style="width:143px;">3013</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Trypsin-Versene mixture</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-161E</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP231-100</td>
			<td>Combustible liquid. Can cause skin, eye and respiratory tract irritation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryogenic vials</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430489</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">PLAGA</td>
			<td style="width:139px;">Purac Biomaterials</td>
			<td style="width:143px;">Purasorb PLG8523</td>
			<td>Store at -800C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCPS disks</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">12-545-82</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Dichloromethane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC36423-0010</td>
			<td>Possible cancer hazard. Store in a dry, cool place.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Bytac paper</td>
			<td style="width:139px;">Saint gobin performance plastics</td>
			<td style="width:143px;">1420652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintillation vial</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>03-339-21G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A7906</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">mouse Anti-&#946;-actin antibody (10 Ab)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A5441</td>
			<td>Store at -200C</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">goat-anti-mouse antibody (20 Ab)</td>
			<td style="width:139px;">Cell Signaling</td>
			<td style="width:143px;">4408</td>
			<td>Store at -200C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Hoechst dye</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">14530</td>
			<td>Store at -200C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP229-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP2547-1</td>
			<td>Toxic by inhalation and if swallowed. Causes burns by all exposure routes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexamethyldisilazane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC43085-1000</td>
			<td>Flammable liquid and vapor. Causes burns by all exposure routes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile Scissors</td>
			<td style="width:139px;">McKesson</td>
			<td style="width:143px;">25-716</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:139px;">Eppendorf</td>
			<td style="width:143px;">5804R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light Microscope</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:143px;">CK40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water Bath</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:143px;">2845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:139px;">Labnet&#160;</td>
			<td style="width:143px;">VX-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Petri plates</td>
			<td style="width:139px;">fisher Scientific</td>
			<td style="width:143px;">S31473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acu-Punch</td>
			<td style="width:139px;">Acuderm Inc.</td>
			<td style="width:143px;">P1225</td>
			<td>Acu-Punch was used to cut 12mm disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cacodylate buffer</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">97068</td>
			<td>Flammable liquid, carcinogen and irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium tetraoxide</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">201030</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">T8787</td>
			<td>Harmful if swallowed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:139px;">Decon Labs</td>
			<td style="width:143px;">2705</td>
			<td>Keep away from heat, sparks, flame and other form of ignition.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">General/regional Anesthesia</td>
			<td style="width:139px;">Amphastar pharmaceuticals</td>
			<td style="width:143px;"></td>
			<td>1% Lidocaine, 0.25% Bupivacaine,Anesthetic agents for induction and maintenance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotics</td>
			<td style="width:139px;">Hospira</td>
			<td>0409-0805-01</td>
			<td>Ancef 1g i.v.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arthroscopy trocar</td>
			<td style="width:139px;">Smith and Nephew</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arthroscopy Camera</td>
			<td style="width:139px;">Smith and Nephew</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arthroscopic grasper and bitter</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4.5mm shaver</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Interference Screws</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td>stainless steel screws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sputter Coater</td>
			<td style="width:139px;">Polaron</td>
			<td style="width:143px;">E5400</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications

ACLに損傷がアクティブの個体において一般的に発生する問題です。機能性と安定性を損なう生体力学的な欠陥は、この関節膝靭帯リードのさえ部分的な涙。熱修正、シングルバンドルの修復、完全な復興、およびネイティブ靭帯の損傷部分の再構築：部分的なACLの涙の治療のための現在のオプションは保存療法、保守的な経営陣からのような複数の外科オプションの範囲。いくつかの研究は、もしあれば、一貫して優れていると管理のための任意の単一の方法を実証しており、多くの場合、患者は、永続的な不安定性および他の併存疾患を実証し続ける。 本研究の目的は、部分的に引き裂かれたACLの修復に実施することができる組織工学パッチの開発に利用するための潜在的な細胞源を同定することである。新規プロトコルはpatieに由来する細胞の拡大のために開発されましたACL再建を受けてNTS。細胞を単離するために、ACL再建中に得られたミンチHACL組織をコラゲナーゼ溶液中で消化した。膨張は、10％ウシ胎児血清（FBS）を補充したDMEM/F12培地および1％ペニシリン/ストレプトマイシン（P / S）を用いて行った。次いで、細胞をDMSO、FBSおよび拡張培地からなる凍結培地中に-80℃で、または液体窒素中で保存した。解凍後、HACL由来細胞は、次いで、組織工学的足場、PLAGA（ポリ乳酸 - コ - グリコール酸）および対照組織培養ポリスチレン（TCPS）上に播種した。 7日後、SEMを制御TCPSに対するPLAGAに対する細胞接着性を比較した。細胞形態は、免疫蛍光染色を用いて評価した。 SEM（走査型電子顕微鏡）顕微鏡写真は、細胞が成長し、PLAGAおよびTCPS両面に付着し、7日目で全面にコンフルエントことを明らかにした。免疫蛍光染色は、通常、非ストレス形態学を示した両面にアルミニウムパターン。この手法は、ACLの再生·復興への応用が期待されている。

外科用回収、組織消化し ​​、ヒト前十字靭帯（HACL）由来細胞の単離のためのワーキングモデルを図1に示す。外植片から移行し、T-25フラスコに付着した細胞を。これらの細胞を3日間培養し、光学顕微鏡（ 図2）の下で可視化した。コンフルエントな単層を、7日目までに得た。健康な、生存細胞の存在はHACL由来細胞の正常な取得および培養を示す。 <p class="jove_c...

Watch this Scientific Journal Video about Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications at JoVE.com

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

前十字靭帯（ACL）の部分的な涙を修復するためのパッチなど、将来のアプリケーションでは、人間のACL由来の細胞をin vitroで増殖し、組織工学足場上に成長させ、再建手続きの際に得られた組織から単離した。細胞接着および形態は、その後、足場表面上に生体適合性を確認した。

外科用回収、単離と<em&gt;インビトロ</em&gt;組織工学アプリケーションのためのヒト前十字靭帯由来細胞の拡大

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

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Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

11.4K ビュー.  Southern Illinois University School of Medicine. 前十字靭帯（ACL）の部分的な涙を修復するためのパッチなど、将来のアプリケーションでは、人間のACL由来の細胞をin vitroで増殖し、組織工学足場上に成長させ、再建手続きの際に得られた組織から単離した。細胞接着および形態は、その後、足場表面上に生体適合性を確認した。 

ビデオ: Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

人間の3次元の組織工学<em&gt; in vitroで</em&gt;腫瘍テストシステム

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

生物工学

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

エンジニアード前十字靭帯移植片の評価のための無胸腺ラットモデル

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

の応用<em&gt;インビボ</em組織工学骨格筋の修復を評価するためのラット前脛骨の&gt;機能テスト

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

組織工学的応用の制御可能な形態の三次元 Poly(ε-caprolactone) 足場のエレクトロスピニング記述を溶かす

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人間歯槽骨膜から骨膜由来細胞の骨形成促進活性に及ぼすビタミン D の間葉系幹細胞の分離

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

組織工学用植物組織の Decellularization の 2 つの方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

in vitroヒト線維芽細胞増殖を促進するための自己多血小板血漿の産生

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

オープングローブ損傷と治療性能を追跡するための前セグメント臓器培養プラットフォーム

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

スクリーニング用途のための線維芽細胞由来ヒト工学的結合組織

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...