Name: electrically conductive scaffold to modulate and deliver stem cells
Uploaded: 2018-04-13T13:30:00.000+00:00
Duration: 5 min 49 s
Description: Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

QRT PCR 機の使用ありがとう博士カティ andreasson 間違いなく天才 (神経内科・脳神経科学、スタンフォード大学)。仕事は (P.M.G.) に健康補助金 K08NS098876 の国立研究所およびスタンフォード大学医学部学部長員 (体臭) にによって支えられました。

すべての幹細胞と動物の手続きは研究所動物愛護 (SCRO 616、APLAC 31909) のスタンフォード大学の管理パネル、スタンフォード大学の幹細胞研究管理委員会によって承認されました。
1. ガラス基板エッチング
<ol><li>インジウム錫酸化物 (ITO) の準備-ガラスの導電面にマスキング剤を配置することによって覆われ、ガラスのスライド。 注: ほとんどの市販の透明テープは、マスキング剤として使用できます。<ol>
<li>刃を使用して余分なマスクを削除、ガラスに覆われた目的の最終的な伊藤設計の形状だけを残してします。</li>
	</ol>
</li>
	<li>硝酸の 5% (v/v) と 45% (v/v) エッチング ソリューションを準備するヒューム フード内ガラス ビーカーに塩酸を組み合わせます。<ol>
<li>70 ° C でエッチング液の温度を維持するのにホット プレートと温度計を使用します。</li>
	</ol>
</li>
	<li>エッチング液 70 ° C に達すると、過剰の ITO 層を取除くために 2 分のためのソリューションにマスク ITO ガラス スライドを配置します。 注: テープ マスキングは酸溶液への暴露から ITO 層を保護します。</li>
	<li>スライドをソリューションから削除し、脱イオン (DI) H2o. でそれをすすいでください。</li>
	<li>マルチメータを使用して残りの伊藤が維持されていることを確認するマスクおよび測定抵抗を慎重に取り外します。目的のエッチング領域のリードアウトは、約 0 Ω をする必要があります。</li>
</ol>2. ピロール溶液の調製
<ol><li>1000 mL のガラス瓶の中・ ディ ・ H2o. の 600 mL のドデシルベンゼンスルホン酸ナトリウム (NaDBS) の 70 g を溶かす</li>
	<li>NaDBS が解散した後 14 mL 0.2 M ピロールと・ ディ ・ H2O の mL の 386 をソリューションに追加します。</li>
	<li>アルミ箔とソリューションをカバーし、4 ° C で保管</li>
</ol>3. めっきガラス基板上ヘのポリピロール
<ol><li>NaDBS は完全に再溶解されるまでは、部屋の温度にピロール解決を暖めます。 注: これは、約 15 〜 20 分かかります。</li>
	<li>ピロール溶液 25 mL をビーカーに注ぐ。</li>
	<li>マルチメータにエッチングのガラス基板を接続し、ピロール ソリューションにスライドが水没します。<ol>
<li>0 Ω 抵抗側がプラチナ メッシュ参照電極に向かって直面していることを確認します。</li>
	</ol>
</li>
	<li>PPy マルチメータ (電流 3 mA/cm2で 4 時間制御) を用いた ITO 表面にめっきを開始する電流を適用します。</li>
	<li>デジタルマルチメータと洗って電気めっき-PPy ・ ディ ・ H2O 残留界面活性剤を削除するから ITO ガラスを削除します。</li>
	<li>かみそりの刃を使用して ITO ガラスからめっき PPy プレートを注意深く取り外します。<ol>
<li>常温 PPy プレートを格納します。</li>
	</ol>
</li>
</ol>4. ポリジメチルシロキサン (PDMS) の準備
<ol><li>ヘラと計量皿を使用して、基本エージェントの 18 g を混ぜて硬化剤 2 g と 2 つ 10 cm × 8 cm ガラス金型に流し込みます。</li>
	<li>15 〜 20 分の真空チャンバーを使用して金型から泡を削除します。</li>
	<li>3 h の 70 ° C のオーブンで金型を配置します。</li>
	<li>いったん冷却、金型から、PDMS を削除するのに平らなヘラを使用します。</li>
</ol>5.体外刺激チャンバーの作製
<ol><li>オートクレーブ 1 時間 120 ° C で金属板 (WxL、2.5 cm × 12.5 cm) とフロー バルブします。</li>
	<li>チェンバー スライドをテンプレートとして使用して、PDMS の 2 つの部分をカットします。 注: PDMS のワンピースは、セル室の細胞部屋から 2 つの隣接する正方形の排気切替器との境界として機能します。もうひとつ、PDMS 商工会議所の境界の概要であります。<ol>
<li>カット内部をセル商工会議所、それは正方形形、セル室の図形と一致します。PDMS の両方の部分の全体の形状は長方形、チェンバー スライドのと一致することを確認します。</li>
	</ol>
</li>
	<li>層材料のトップに下から次のよう: (1) メタル プレート、 (切り抜き)、なし (2) PDMS (3) PPy プレート (PDMS に垂直)、 (切り欠き) PDMS (4) と (5) セルの商工会議所。</li>
	<li>一度完全に整列装置を一緒にクランプします。</li>
	<li>金属線の 2 つの 60 cm 部分をカットし、商工会議所の外側から突き出ている PPy 板の両端に 1 本のワイヤーを接続する銀ペーストを使用します。その線がインキュベーターの外側に装置を拡張するのに十分な長さを確認します。</li>
	<li>銀ペーストの硬化後、強化し、エポキシとワイヤーの接続をシールします。<ol>
<li>応用分野は、各商工会議所で同じであることを確認するテスターを使用して抵抗を記録します。</li>
		<li>注入、削除線;細胞培養チャンバーをアンクランプし、PDMS し、それらを導電性の足場から分離します。注入された足場の寸法は、約 1 × 3 × 0.25 mm です。</li>
	</ol>
</li>
</ol>6. ひと神経前駆細胞 (hNPCs) PPy 上のめっき
<ol><li>2 時間の紫外線の下で組み立て室を滅菌します。<ol>
<li>1 時間後回転組み立て室 90 °。</li>
	</ol>
</li>
	<li>滅菌後、PPy コーティング基板の 800 μ L でチャンバーの下部の表面をコート、1 h の 5% CO2で 37 ° C でインキュベーターで PPy プレート上を固めるため基板を許可します。</li>
	<li>1 時間後の部屋から上澄みを除去し、ウェルあたり DPBS + Ca、Mg の 1 mL で優しくすすいでください。 メモ: は、基材の剥離の原因になります、力強く PPy の表面を洗浄しないでください。</li>
	<li>新鮮な携帯メディアを使用して、機械的に穏やかなピペッティングで組織培養プレートからチャンバー用培養細胞を切り離して考えます。 注: セルは 90-100% 解離に合流をする必要があります。</li>
	<li>8000 × g で 5 分間遠心分離を用いた hNPC ペレットを収集します。</li>
	<li>診断を使用して、カウント、100,000 セル/cm2のボリュームに細胞を再懸濁します。</li>
	<li>各商工会議所よく (100,000 セル/cm2メディアの 1 ml) に 100,000 細胞をプレートします。</li>
	<li>5% で 37 ° C でインキュベーターに部屋を戻る CO2.
</li>
</ol>7. hNPCs の刺激
<ol><li>1 日播種後、チャンバーに細胞は、細胞に電気刺激を適用するのに波形発生器を使用します。</li>
	<li>刺激の前に各商工会議所からも古いメディアを削除し、新鮮なメディアの 800 μ L で補充します。</li>
	<li>メディアが変更された後チャンバーをインキュベーターに配置、インキュベーター、外ワイヤーを拡張し、波形発生器に接続</li>
	<li>注 mV、100 Hz と 1 h で方形波信号を細胞に刺激を適用されます。</li>
	<li>、刺激後ワイヤーを取り外して 5% CO2と 37 ° C で設定インキュベーターの別の日の両院を孵化させなさい。</li>
	<li>セル実行可能性および量的なリアルタイムのポリメラーゼの連鎖反応 (qRT PCR) 次の製造元のプロトコルを含む後続の生物学的解析を実行します。</li>
</ol>8. PPy体内注入
<ol><li>T 細胞欠損の遠位中大脳動脈 (dMCA) 閉塞ストローク モデルを実行として成人男性裸ラット (NIH RNU 230 ± 30 g) は前述2。注入 1 週間ポスト dMCA オクルー ジョンを実行します。</li>
	<li>手術の前日は、ケージ水ラットにアンピシリン (1 mg/mL) を与えます。</li>
	<li>使用して誘導室にラットを麻酔イソフルラン吸入による投与の 0.5 - 3 %mg/kg。</li>
	<li>つま先ピンチ反射応答の欠如によって、ラットの anesthetization を確認してください。<ol>
<li>動物は、麻酔下では、その膜の色、呼吸パターン、および直腸温 15 分毎に監視を継続します。</li>
	</ol>
</li>
	<li>Anesthetization を確認した後は、乾燥を防ぐためにラットの目に人工涙液を適用します。</li>
	<li>無菌が滅菌手袋、滅菌手術用ドレープ13を使用して、この手順の間維持されることを確認します。滅菌フィールド内で無菌手術器械を保ちます。</li>
	<li>麻酔下ラットですが、左の皮質上頭をドリルします。硬膜を開きます。<ol>
<li>マイクロ薄い手術用の針を使用して脳から硬膜を削除します。</li>
	</ol>
</li>
	<li>インプラント システムの in vitroラット大脳皮質から導電性足場。 注: 実験の日 3 hNPC めっき後体外から足場を注入しました。<ol>
<li>脳卒中病変の内側半影野で主にインプラントを配置します。</li>
	</ol>
</li>
	<li>足場を配置すると後、は、皮膚縫合の動きを防ぐためにインプラントの上 surgicel を置きます。</li>
	<li>縫合傷がシャット ダウンし、ブプレノルフィン SR 定位手術と dMCA の閉塞に伴う痛みを管理するための 1 mg/kg の用量でラット皮下注入します。</li>
	<li>意識を取り戻すと、回復ケージにラットを配置します。<ol>
<li>動物が離れないで意識ではありません。</li>
		<li>それは完全に意識を得ているまでは、他の人と動物を置かないでください。</li>
	</ol>
</li>
	<li>体重減少、減少食品/水の摂取量、削減グルーミング/ボサボサ コート、自発運動の減少/増加、異常姿勢、脱水/スキン ・ テンティング、くぼんだ目、隠れ、自傷などの痛みの兆候を毎日動物を監視します。急速な呼吸、開いて口呼吸、けいれん、震え、震えと発声。<ol>
<li>食べる、飲む、グルーミング、体重; 表示されるまで毎日動物を監視します。毎週体重増加の後。</li>
	</ol>
</li>
	<li>飲んで食べてない、表示される任意の動物に CO2吸入と (のような動物が正常な酸素供給のポスト CO2吸入による復活しないようにする) euthanization の二次確認を介して初期の euthanization が発生して初期手術後の体重苦痛に表示されます。または運動皮質の赤字を予想よりも大きいため行動のテストを完了することができませんが表示されます。</li>
</ol>

<ol>
	<li>Moskowitz, M. A., Lo, E. H., Iadecola, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Science+of+Stroke:+Mechanisms+in+Search+of+Treatments.">The Science of Stroke: Mechanisms in Search of Treatments.</a> Neuron. 67 (2), 181-198 (2010).</li><li>George, P. M., 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=Electrical+Preconditioning+of+Stem+Cells+with+a+Conductive+Polymer+Scaffold+Enhances+Stroke+Recovery.">Electrical Preconditioning of Stem Cells with a Conductive Polymer Scaffold Enhances Stroke Recovery.</a> Biomaterials. 142, 31-40 (2017).</li><li>Steinberg, G. 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=Clinical+Outcomes+of+Transplanted+Modified+Bone+Marrow-Derived+Mesenchymal+Stem+Cells+in+Stroke:+A+Phase+1/2a+Study.">Clinical Outcomes of Transplanted Modified Bone Marrow-Derived Mesenchymal Stem Cells in Stroke: A Phase 1/2a Study.</a> Stroke. 47 (7), 1817-1824 (2016).</li><li>George, P. M., Steinberg, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Stroke+Therapeutics:+Unraveling+Stroke+Pathophysiology+and+Its+Impact+on+Clinical+Treatments.">Novel Stroke Therapeutics: Unraveling Stroke Pathophysiology and Its Impact on Clinical Treatments.</a> Neuron. 87 (2), 297-309 (2015).</li><li>Schabitz, W. R., 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=Intravenous+Brain-Derived+Neurotrophic+Factor+Enhances+Poststroke+Sensorimotor+Recovery+and+Stimulates+Neurogenesis.">Intravenous Brain-Derived Neurotrophic Factor Enhances Poststroke Sensorimotor Recovery and Stimulates Neurogenesis.</a> Stroke. 38 (7), 2165-2172 (2007).</li><li>Liauw, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondins+1+and+2+Are+Necessary+For+Synaptic+Plasticity+and+Functional+Recovery+After+Stroke.">Thrombospondins 1 and 2 Are Necessary For Synaptic Plasticity and Functional Recovery After Stroke.</a> J Cereb Blood Flow Metab. 28 (10), 1722-1732 (2008).</li><li>Cantu, D. A., Hematti, P., Kao, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Encapsulating+Biomaterial+Regulates+Mesenchymal+Stromal/Stem+Cell+Differentiation+and+Macrophage+Immunophenotype.">Cell Encapsulating Biomaterial Regulates Mesenchymal Stromal/Stem Cell Differentiation and Macrophage Immunophenotype.</a> Stem Cells Translational Medicine. 1 (10), 740-749 (2012).</li><li>Chaudhuri, O., 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=Hydrogels+with+Tunable+Stress+Relaxation+Regulate+Stem+Cell+Fate+and+Activity.">Hydrogels with Tunable Stress Relaxation Regulate Stem Cell Fate and Activity.</a> Nat Mater. 15 (3), 326-334 (2016).</li><li>Ballios, B. G., 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+Hyaluronan-Based+Injectable+Hydrogel+Improves+the+Survival+and+Integration+of+Stem+Cell+Progeny+following+Transplantation.">A Hyaluronan-Based Injectable Hydrogel Improves the Survival and Integration of Stem Cell Progeny following Transplantation.</a> Stem Cell Reports. 4 (6), 1031-1045 (2015).</li><li>Mothe, A. J., Tam, R. Y., Zahir, T., Tator, C. H., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+the+Injured+Spinal+Cord+by+Transplantation+of+Neural+Stem+Cells+in+a+hyaluronan-based+hydrogel.">Repair of the Injured Spinal Cord by Transplantation of Neural Stem Cells in a hyaluronan-based hydrogel.</a> Biomaterials. 34 (15), 3775-3783 (2013).</li><li>Saini, M., Singh, Y., Arora, P., Arora, V., Jain, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implant+Biomaterials:+A+Comprehensive+Review.">Implant Biomaterials: A Comprehensive Review.</a> World J Clin Cases. 3 (1), 52-57 (2015).</li><li>Huang, Y., Li, Y., Chen, J., Zhou, H., Tan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+Stimulation+Elicits+Neural+Stem+Cells+Activation:+New+Perspectives+in+CNS+Repair.">Electrical Stimulation Elicits Neural Stem Cells Activation: New Perspectives in CNS Repair.</a> Front Hum Neurosci. 9, (2015).</li><li>Pritchett-Corning, K. R., Mulder, G. B., Luo, Y., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Rodent+Surgery+for+the+New+Surgeon.">Principles of Rodent Surgery for the New Surgeon.</a> J Vis Exp. (47), (2011).</li><li>Abdelwahid, 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=Stem+Cell+Death+and+Survival+in+Heart+Regeneration+and+Repair.">Stem Cell Death and Survival in Heart Regeneration and Repair.</a> Apoptosis. 21 (3), 252-268 (2016).</li><li>Wang, X., 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+Clinical+Status+of+Stem+Cell+Therapy+for+Ischemic+Cardiomyopathy.">The Clinical Status of Stem Cell Therapy for Ischemic Cardiomyopathy.</a> Stem Cells Int. 2015, (2015).</li><li>George, P. M., 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=Fabrication+and+Biocompatibility+of+Polypyrrole+Implants+Suitable+for+Neural+Prosthetics.">Fabrication and Biocompatibility of Polypyrrole Implants Suitable for Neural Prosthetics.</a> Biomaterials. 26 (17), 3511-3519 (2005).</li><li>Aravamudan, B., Thompson, M., Pabelick, C., Prakash, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-Derived+Neurotrophic+Factor+Induces+Proliferation+of+Human+Airway+Smooth+Muscle+Cells.">Brain-Derived Neurotrophic Factor Induces Proliferation of Human Airway Smooth Muscle Cells.</a> J Cell Mol Med. 16 (4), 812-823 (2012).</li><li>Lopes, N., 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=Thrombospondin+2+Regulates+Cell+Proliferation+Induced+by+Rac1+Redox-Dependent+Signaling.">Thrombospondin 2 Regulates Cell Proliferation Induced by Rac1 Redox-Dependent Signaling.</a> Mol Cell Biol. 23 (15), 5401-5408 (2003).</li><li>Ploughman, M., 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=Brain-Derived+Neurotrophic+Factor+Contributes+to+Recovery+of+Skilled+Reaching+After+Focal+Ischemia+in+Rats.">Brain-Derived Neurotrophic Factor Contributes to Recovery of Skilled Reaching After Focal Ischemia in Rats.</a> Stroke. 40 (4), 1490-1495 (2009).</li><li>Baker, E. W., 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=Induced+Pluripotent+Stem+Cell-Derived+Neural+Stem+Cell+Therapy+Enhances+Recovery+in+an+Ischemic+Stroke+Pig+Model.">Induced Pluripotent Stem Cell-Derived Neural Stem Cell Therapy Enhances Recovery in an Ischemic Stroke Pig Model.</a> Sci Rep. 7 (1), 10075 (2017).</li><li>Bacigaluppi, M., 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=Neural+Stem+Cell+Transplantation+Induces+Stroke+Recovery+by+Upregulating+Glutamate+Transporter+GLT-1+in+Astrocytes.">Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes.</a> J Neurosci. 36 (41), 10529-10544 (2016).</li></ol>

著者はこの仕事を宣言する利害の対立があります。

成長の証拠として新規ストローク療法幹細胞の約束を実証しています。この約束は、少なくとも 40 進行中または完了した臨床試験とベッドサイドに幹細胞治療を進めるための主要な努力になりました。ストローク病理学では、幹細胞療法に向いている急性侮辱後回復を防止する神経変性プロセスがないためユニークな神経疾患を提供しています。幹細胞が強化された脳卒中の回復の正確なメ...

ストロークは、世界の死の 2 番目の主要な原因とアメリカ合衆国の死の 5 番目の主要な原因です。にもかかわらず、これらの高死亡率、脳卒中の回復のための治療現在無し医療選択肢の現在利用可能な1課題のまま。うちだけ 40 利用幹細胞の虚血性脳卒中を扱う 300 の臨床試験について現在あります。前の研究は、幹細胞治療にストローク修理2,3に有益な効果があることを示しています。脳由来神経栄養因子 (BDNF) やトロンボスポンジン 1 (THBS-1) 移植ひと神経前駆細胞 (hNPCs) から放出されたなどのパラクライン因子の増加に関連付けられているメカニズムを通して機能の回復機能の向上が示されています。シナプス形成、血管新生、樹状突起分岐、新しい軸索投射だけでなく、免疫系4,5,6を調節すること。ただし、幹細胞の最適な配信方法は、とらえどころのないままです。
脳に成功した幹細胞配信課題のまま。現在、幹細胞を提供する注射ハイドロゲルと高分子足場システムが導入されています。移植時に幹細胞を保護し、宿主の炎症反応と低酸素の条件7,8,9を含む過酷な脳卒中後の環境からの保護を提供するこれらの送付方法,10します。 ただし、最も一般的に使用される材料は、不活性細胞11の連続変調方式 (すなわち、電気刺激) の使用を制限します。電気刺激は分化、イオン チャネル密度、および幹細胞12の神経突起伸展に影響を与えるです。不活性ポリマーと比較して、導電性高分子は電気刺激と幹細胞2の操作を現在可能を運ぶことができます。ただし、電気刺激、(すなわち、 BDNF および THBS 1) 神経栄養因子の遊離を調節する正確なメカニズムはまだ完全に検討されます。
このプロトコルでは、導電性高分子の足場は、体外の電気刺激は、ポリピロール (PPy) から成る細胞培養系を構築する手順を示します。細胞培養系を作製方法により遷延皮質に幹細胞シード足場の後続の注入が可能です。このシステムのため我々 電気的前提、裏付け足場に幹細胞注入前に短期間のため。次の電気刺激は、低侵襲による頭蓋内細胞を運ぶ導電性高分子足場は正常に注入しました。

<table><tbody><tr><td>FGF-Basic</td><td>Invitrogen</td><td>CTP0261</td><td>20 ng/mL for working media</td></tr><tr><td>Matrigel</td><td>Corning</td><td>cb40234a</td><td>1:200 dilution</td></tr><tr><td>LIF Protein, Recom. Hum. (10 &amp;micro;g/mL)</td><td>EMD Millipore</td><td>LIF1010</td><td>10 ng/mL for working media</td></tr><tr><td>Sylgard 184 silicone 3.9 kg</td><td>Fisherbrand</td><td>NC0162601</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisherbrand</td><td>SA56-4</td><td></td></tr><tr><td>Nitric Acid Concentrate (Certified) ACS, Fisher Chemical</td><td>Fisherbrand</td><td>SA95</td><td></td></tr><tr><td>ITO Glass</td><td>Delta Technologies</td><td>CG-40IN-0115</td><td></td></tr><tr><td>Sodium dodecylbenzenesulfonate</td><td>Sigma</td><td>289957-1KG</td><td></td></tr><tr><td>Pyrrole</td><td>Sigma</td><td>131709-500ML</td><td>Protect pyrrole solution from light and room temperature</td></tr><tr><td>8 well glass slide chambers</td><td>Thermo Sci Nuc&amp;nbsp;</td><td>125658</td><td>Detach the cell chamber and keep it under sterilized conditions</td></tr><tr><td>Flat-Surface Bracket, 3&quot;x1&quot;</td><td>McMaster-Carr&amp;nbsp;</td><td>1030A4</td><td></td></tr><tr><td>TWO PART SILVER PAINT 14G</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>1264214</td><td>Mix two parts (1:1) in plastic plate</td></tr><tr><td>DPBS, 1x, with Ca and Mg, No Phenol Red</td><td>Genesse&amp;nbsp;</td><td>25-508C</td><td></td></tr><tr><td>AB2 ArunA Neural Cell Culture Media Kit</td><td>Aruna Biomedical&amp;nbsp;</td><td>ABNS7013.2</td><td></td></tr><tr><td>hNP1 Human Neural Progenitor Expansion Kit</td><td>Aruna Biomedical&amp;nbsp;</td><td>&amp;nbsp;hNP7013.1</td><td></td></tr><tr><td>Noncontact Flow-Adjustment Valve, Nickel-Plated Brass, for 3/32&quot; to 5/8&quot; Tube OD</td><td>McMaster-Carr&amp;nbsp;</td><td>5330K22</td><td></td></tr><tr><td>Multimeter</td><td>Keysight&amp;nbsp;</td><td>E3641A</td><td></td></tr><tr><td>Wavefoam generator</td><td>Keysight</td><td>33210A-10MHz</td><td></td></tr><tr><td>Pt meshes</td><td>Sigma-Aldrich&amp;nbsp;</td><td>298107-425MG</td><td>Reference electrode with dimensions, 1x1 cm</td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>L3224</td><td></td></tr><tr><td>BDNF</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs02718934_s1</td><td></td></tr><tr><td>THBS1</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs00962908_m1</td><td></td></tr><tr><td>GAPDH</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs02758991_g1</td><td></td></tr><tr><td>RNeasy Mini Kit (250)</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>QIAshredder (250)</td><td>Qiagen</td><td>79656</td><td></td></tr><tr><td>RNase-Free DNase Set (50)</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit, 100 x 20 &amp;micro;L rxns</td><td>BIORAD</td><td>1708891</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>4369510</td><td></td></tr><tr><td>7-8 Week Old, male, RNU Rats</td><td>NCI-Frederick</td><td></td><td></td></tr><tr><td>4-0 Ethicon Silk Suture</td><td>eSutures.com</td><td>683G</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>29405</td><td></td></tr><tr><td>V-1 Tabletop Laboratory Animal Anesthesia System</td><td>VetEquip</td><td>901806</td><td></td></tr><tr><td>Surgicel Original Absorbable Hemostat</td><td>Ethicon</td><td>1952</td><td></td></tr><tr><td>Lab Standard Stereotaxic Instrument, Rat</td><td>Stoelting</td><td>51600</td><td></td></tr><tr><td>Kimberly-Clark Professional&amp;nbsp;Safeskin Purple Nitrile Sterile Exam Gloves</td><td>Fisherbrand</td><td>19-063-130</td><td></td></tr><tr><td>Sterile Drape</td><td>Medline</td><td>DYNJSD1092</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Shandon Stainless-Steel Scalpel Blade Handle, Holds No. 20-25 Blades</td><td>Fisherbrand</td><td>53-34</td><td></td></tr><tr><td>Walter Stern Scalpel Blade Series 300</td><td>Fisherbrand</td><td>17-654-456</td><td></td></tr><tr><td>QuantStudio 6 Flex Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>4484642</td><td></td></tr><tr><td>Frazier Micro Dissecting Hook</td><td>Harvard Apparatus</td><td>52-2706</td><td></td></tr></tbody></table>

electrically conductive scaffold to modulate and deliver stem cells

幹細胞療法が治療、エキサイティングなストロークとして浮上しているが、最適な配信方法は不明のまま。マイクロインジェクション法は脳卒中モデルで幹細胞を提供する何十年も使用されてきましたが、この手法は注入前に幹細胞を操作する能力の欠如によって制限されます。本稿では、幹細胞配信用導電性高分子足場の使用法を詳しく説明します。導電性高分子足場を用いた幹細胞の電気刺激は、細胞の生存、炎症反応とシナプスのリモデリングに関与する幹細胞の遺伝子を変更します。電気前処理後は、足場に幹細胞は遠位の中大脳動脈閉塞ラット モデルで頭蓋内移植されます。このプロトコルは、導電性高分子足場経由で幹細胞を操作するための強力な手法をについて説明し、さらに幹細胞を用いた治療法を開発するための新しいツールを作成します。

図 1に示す回路図は、hNPCs および潜在的なダウン ストリーム アプリケーションの電気刺激の全体的なワークフローを表します。幹細胞治療で現在の制限は、幹細胞が炎症や虚血性の条件を含む厳しい移植後環境にさらされていることです。そうこれらの困難な条件は、その治療効果14,15を制限します?...

Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

このプロトコルを記述するように導電性高分子足場の in vitro電気刺激および幹細胞播種の足場を使用しての注入その後体内の幹細胞の播種細胞培養系の作製、低侵襲技術。

導電性足場を調節して幹細胞を提供するには

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

electrically-conductive-scaffold-to-modulate-and-deliver-stem-cells

Research

JoVE Journal

Bioengineering

12.7K ビュー.  Stanford University School of Medicine. このプロトコルを記述するように導電性高分子足場の in vitro電気刺激および幹細胞播種の足場を使用しての注入その後体内の幹細胞の播種細胞培養系の作製、低侵襲技術。

ビデオ: Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

骨再生のための人間の血管周囲の幹細胞の使用

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

生物工学

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

組織工学：層状細胞シートの送達のための多細胞3D足場の構築

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

ポリ（εカプロラクトン）エレクトロ糸にヒト間葉系幹細胞の添付ファイルの最適化

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

繊維組織における階調の電界紡糸ナノファイバー足場

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

軟骨再生のための3次元磁気幹細胞凝集およびバイオリアクター成熟

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

細胞の Cardiomyogenic の可能性を高めるに同時電気的・機械的刺激

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

建設と質間葉系幹細胞の骨分化を強化するため電気刺激室の使用

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

シュワン細胞表現型仕様を評価するための細胞外マトリックス微小環境の調製

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

細胞電微小環境の有限元素モデリング

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

慢性創傷管理のためのコアシース3Dバイオプリントスキャフォールドを使用した細胞外小胞送達の最適化

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol ...