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>

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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。

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			<td height="40" style="height:40px;width:213px;">FGF-Basic</td>
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			<td style="width:188px;">10 ng/mL for working media</td>
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			<td style="width:188px;">Protect pyrrole solution from light and room temperature</td>
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			<td style="width:188px;">Detach the cell chamber and keep it under sterilized conditions</td>
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			<td style="width:119px;">1264214</td>
			<td style="width:188px;">Mix two parts (1:1) in plastic plate</td>
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			<td height="40" style="height:40px;width:213px;">DPBS, 1x, with Ca and Mg, No Phenol Red</td>
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			<td style="width:119px;">25-508C</td>
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			<td height="40" style="height:40px;width:213px;">AB2 ArunA Neural Cell Culture Media Kit</td>
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			<td height="40" style="height:40px;width:213px;">hNP1 Human Neural Progenitor Expansion Kit</td>
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			<td style="width:119px;">&#160;hNP7013.1</td>
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			<td height="60" style="height:60px;width:213px;">Noncontact Flow-Adjustment Valve, Nickel-Plated Brass, for 3/32&#34; to 5/8&#34; Tube OD</td>
			<td style="width:103px;">McMaster-Carr&#160;</td>
			<td style="width:119px;">5330K22</td>
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			<td height="21" style="height:21px;width:213px;">Multimeter</td>
			<td style="width:103px;">Keysight&#160;</td>
			<td style="width:119px;">E3641A</td>
			<td style="width:188px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Wavefoam generator</td>
			<td style="width:103px;">Keysight</td>
			<td style="width:119px;">33210A-10MHz</td>
			<td style="width:188px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Pt meshes</td>
			<td style="width:103px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">298107-425MG</td>
			<td style="width:188px;">Reference electrode with dimensions, 1x1 cm</td>
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		<tr height="61">
			<td height="61" style="height:61px;width:213px;">LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">L3224</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">BDNF</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs02718934_s1</td>
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			<td height="61" style="height:61px;">THBS1</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs00962908_m1</td>
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			<td height="61" style="height:61px;width:213px;">GAPDH</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs02758991_g1</td>
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			<td height="21" style="height:21px;width:213px;">RNeasy Mini Kit (250)</td>
			<td style="width:103px;">Qiagen</td>
			<td style="width:119px;">74106</td>
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			<td height="21" style="height:21px;width:213px;">QIAshredder (250)</td>
			<td style="width:103px;">Qiagen</td>
			<td style="width:119px;">79656</td>
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			<td height="21" style="height:21px;width:213px;">RNase-Free DNase Set (50)</td>
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			<td height="41" style="height:41px;width:213px;">iScript cDNA Synthesis Kit, 100 x 20 &#181;L rxns</td>
			<td style="width:103px;">BIORAD</td>
			<td style="width:119px;">1708891</td>
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			<td height="61" style="height:61px;width:213px;">TaqMan Gene Expression Master Mix</td>
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			<td style="width:119px;">4369510</td>
			<td></td>
		</tr>
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			<td height="41" style="height:41px;width:213px;">7-8 Week Old, male, RNU Rats</td>
			<td style="width:103px;">NCI-Frederick</td>
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			<td height="41" style="height:41px;width:213px;">4-0 Ethicon Silk Suture</td>
			<td style="width:103px;">eSutures.com</td>
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			<td style="width:119px;">29405</td>
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			<td style="width:103px;">VetEquip</td>
			<td style="width:119px;">901806</td>
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			<td height="41" style="height:41px;width:213px;">Surgicel Original Absorbable Hemostat</td>
			<td style="width:103px;">Ethicon</td>
			<td style="width:119px;">1952</td>
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		</tr>
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			<td height="40" style="height:40px;width:213px;">Lab Standard Stereotaxic Instrument, Rat</td>
			<td style="width:103px;">Stoelting</td>
			<td style="width:119px;">51600</td>
			<td></td>
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			<td height="80" style="height:80px;width:213px;">Kimberly-Clark Professional&#160;Safeskin Purple Nitrile Sterile Exam Gloves</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">19-063-130</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:213px;">Sterile Drape</td>
			<td style="width:103px;">Medline</td>
			<td style="width:119px;">DYNJSD1092</td>
			<td></td>
		</tr>
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			<td height="80" style="height:80px;width:213px;">Thermo Scientific&#160;Shandon Stainless-Steel Scalpel Blade Handle, Holds No. 20-25 Blades</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">53-34</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Walter Stern Scalpel Blade Series 300</td>
			<td style="width:103px;">Fisherbrand</td>
			<td>17-654-456</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">QuantStudio 6 Flex Real-Time PCR System</td>
			<td style="width:103px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">4484642</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">Frazier Micro Dissecting Hook</td>
			<td style="width:103px;">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

12.4K 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

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

導電性インクの平面および三次元印刷

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

生物工学

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

異なる系統にヒト胚性幹細胞（hESCの）の伝播との差別化のための3Dプラットフォームとしてのアルギン酸マイクロカプセル

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

開発、展開、および<em生体内で&gt;</em&gt;ヒト胚性幹細胞（ヒトES細胞）からヒトNK細胞のモニタリングとや人工多能性幹細胞（性IPSC）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

シングルセルおよび3D閉じ込めにおける単一胚を注文：ハイコンテントスクリーニングのための新しいデバイスを

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

腎組織のプライマリ セグメントと新規スキャフォールド フリー構造技術と定款の

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

成人骨髄由来造血幹細胞が細胞工学磁気ターゲッティングと結合を有効にするにマイクロ Rna の転送のためのプロトコル

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

静止ヒト造血幹細胞を維持する培養法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

導電性足場を調節して幹細胞を提供するには

要約

さらに動画を探す

この動画の章

導電性インクの平面および三次元印刷

のための周期的圧力のバイオリアクターの設計生体外で研究

異なる系統にヒト胚性幹細胞（hESCの）の伝播との差別化のための3Dプラットフォームとしてのアルギン酸マイクロカプセル

開発、展開、および

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

シングルセルおよび3D閉じ込めにおける単一胚を注文：ハイコンテントスクリーニングのための新しいデバイスを

腎組織のプライマリセグメントと新規スキャフォールドフリー構造技術と定款の

成人骨髄由来造血幹細胞が細胞工学磁気ターゲッティングと結合を有効にするにマイクロ Rna の転送のためのプロトコル

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

静止ヒト造血幹細胞を維持する培養法

導電性足場を調節して幹細胞を提供するには

要約

さらに動画を探す

この動画の章

導電性インクの平面および三次元印刷

のための周期的圧力のバイオリアクターの設計生体外で研究

異なる系統にヒト胚性幹細胞（hESCの）の伝播との差別化のための3Dプラットフォームとしてのアルギン酸マイクロカプセル

開発、展開、および

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

シングルセルおよび3D閉じ込めにおける単一胚を注文：ハイコンテントスクリーニングのための新しいデバイスを

腎組織のプライマリ セグメントと新規スキャフォールド フリー構造技術と定款の

成人骨髄由来造血幹細胞が細胞工学磁気ターゲッティングと結合を有効にするにマイクロ Rna の転送のためのプロトコル

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

静止ヒト造血幹細胞を維持する培養法

腎組織のプライマリセグメントと新規スキャフォールドフリー構造技術と定款の