この作品は、NSFC プロジェクト (81522023、81530059、91639113、81772000、81371699、および 81401534) によって財政的に支えられました。

実験動物の使用は動物実験倫理委員会の南海大学によって承認され、ケアと実験動物の使用のためのガイドに従い実施します。
1. エレクトロスピニング PCL 移植片の作製
注: ここで、エレクトロスピニング法は、人工血管を作製する利用されました。
<ol><li>それぞれ、クロロホルム、メタノールの混合物で PCL を溶解することにより 25 wt % と 10 の wt % の PCL ソリューションを準備 (1:5 容積比) 12 時間 (RT) 室温で。</li>
	<li>10 mL ガラス製注射器に PCL ソリューションを読み込みます。</li>
	<li>21 G 針とシリンジを配置します。</li>
	<li>コレクション楽器にステンレス製マンドレル (長さ 25 cm、直径 2 mm) を配置します。</li>
	<li>25 wt % の PCL ソリューション、コレクターに針先端から 17 cm の作動距離、8 mL/h の流量と 11 の電圧を使用して厚い繊維移植、エレクトロスピニング パラメーターとして kV。シンナー-光ファイバー移植、10 wt %、コレクターに針の先端から 20 cm の作動距離、2 mL/h の流量および 18 の電圧の PCL ソリューションを使用エレクトロスピニング パラメーターとして kV。</li>
	<li>残留溶媒を除去するため一晩に真空で得られた移植が置かれることを確認します。手続きの前にすべての機器を消毒し、全体の無菌技術を維持します。</li>
	<li>注入前に 30 分の 75% エタノール 10 mL でそれらを浸すと、一晩で露出紫外線にグラフトを消毒します。</li>
	<li>繊維と細孔のサイズ測定: 走査電子顕微鏡 (SEM) 像に基づく ImageJ ソフトウェアを使用して平均繊維径を計算。</li>
	<li>機械足場のテスト:<ol>
<li>かみそりの刃を使用して長さで 3 mm セクションに鋼管足場をカットします。マイクロメータを使用して足場の厚さを測定します。</li>
		<li>100 名の負荷容量を持つ引張試験機に鋼管足場を配置します。</li>
		<li>破裂まで 1 mm 間クランプと縦 10 mm/分の速度で引っ張り足場をクランプします。引張強さと伸びの究極を測定します。初期の線形領域からヤング率を計算する (5% 歪み最大) 応力-ひずみ曲線の。</li>
	</ol>
</li>
</ol>2. ラット大動脈移植モデル
注: すべての材料、手術で使用される楽器は、滅菌です。外科の間に演算子がガーゼのマスクと感染症を避けるために滅菌手袋を着ていることを確認します。部屋の温度は動物の体温を維持するために 27-30 ° C に保たれてを確認します。鎮痛に関するローカル IACUC ガイドラインに従います。
<ol><li>血管の受信者として 240 270 グラムの重量を量る男性スプレイグ ラットを使用します。ラットは手術前に、24 時間絶食して、確認します。24 時間絶食ラットの目的は空腸内の糞便、十分に地平線を広げるオペレーターの。</li>
	<li>ラットの首の後ろをつかんで、その頭を保つ springe 針を下腹部の腹腔内に挿入します。腹腔内投与によって抱水クロラール (330 mg/kg) と麻酔ラットを誘発します。</li>
	<li>ラットが筋肉と安定した呼吸を緩和して確保することによって適切な anesthetization を確認します。手術顕微鏡下でラットを仰臥位で配置します。</li>
	<li>麻酔中の乾燥を防ぐため目に眼獣医のワセリン軟膏を適用します。手術前に尾静脈注射によってヘパリン生理食塩液 (50 UI/mL) 抗凝固療法 (100 UI/kg) を管理します。</li>
	<li>かみそりの刃を使用して前方の腹部壁の毛を剃り落とすし、ヨード溶液および医療アルコール溶液を使用して肌をきれい。</li>
	<li>外科はさみで正中開腹切開を行うと確実に約 4-5 cm 長く、切開し、腹腔内を公開します。</li>
	<li>撤回し、優先的に食塩水で湿らせたガーゼで腸をラップします。</li>
	<li>腹部大動脈を慎重に分析します。</li>
	<li>識別し、9-0 モノフィラメント ナイロン縫合糸を使用してすべての小さな枝を結紮します。</li>
	<li>孤立した部分をクランプ (長さ 1 cm まで) の 2 つの血管クランプを用いた大動脈。大動脈は、20-30 分の固定に残ることができます。</li>
	<li>吻合部のサイトを作成するマイクロはさみを使用して 2 つのクランプの間に腹部大動脈を縦断面します。</li>
	<li>ヘパリン生理食塩水 (50 UI/mL) ソリューションを使用して残血を削除する大動脈の 2 つの端をフラッシュします。</li>
	<li>マイクロはさみを使用して外膜をはがします。</li>
	<li>9-0 モノフィラメント ナイロン縫合糸を使っての 8の字縫合パターンを内径 2 mm とのラットの腹部大動脈に長さ 1 cm グラフトを吻合します。</li>
	<li>まず、9、3、12、および近位側で 6 位置の順序に従って 4 つの吻合を構築し、4 針縫合糸 2 つ間で切り口を吻合します。近位部の縫合を終えた後に、同じ方法で遠位側を縫合します。 注: すべてのステッチがネイティブ側やや移植に埋め込まれていることを確認する必要です。</li>
	<li>近位部のクランプを外し、接木に流れ込む血液を許可する遠位クランプを削除します。</li>
	<li>滅菌綿球または小さなガーゼを使用して出血を止めるため縫合糸の末端を押してください。止血まで約 3 分間押します。</li>
	<li>腹腔内に腸を返します。</li>
	<li>ゲンタマイシン (320 U/mL) 暖かい生理食塩液を用いた腹腔内をフラッシュします。</li>
	<li>筋肉と皮膚の層でそれぞれ 3-0 ナイロン縫合糸を使用して腹壁を縫います。</li>
	<li>清潔で乾燥したケージにラットを置き、動物の体の温度を維持するためにケージの下で加熱パッドを入れてください麻酔から回復するラットの待ちます。それは胸骨の横臥を維持するために十分な意識を取り戻したまで動物に出席します。</li>
	<li>後意識を取り戻すことは、食品と水で単一のケージでラットを置きます。手術後の感染を防ぐために傷口にヨウ素を適用します。それが完全に回復するまで、他の動物の会社にネズミを返します。</li>
	<li>あらかじめ決められた時点で制度のガイドラインによるとラットを安楽死させます。</li>
</ol>

<ol>
	<li>Coombs, K. E., Leonard, A. T., Rush, M. N., Santistevan, D. A., Hedberg-Dirk, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+effect+of+material+stiffness+on+valvular+interstitial+cell+differentiation.">Isolated effect of material stiffness on valvular interstitial cell differentiation.</a> J Biomed Mater Res A. 105 (1), 51-61 (2017).</li><li>Zhang, L., 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+sandwich+tubular+scaffold+derived+from+chitosan+for+blood+vessel+tissue+engineering.">A sandwich tubular scaffold derived from chitosan for blood vessel tissue engineering.</a> J Biomed Mater Res A. 77 (2), 277-284 (2006).</li><li>Thottappillil, N., Nair, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+in+vascular+regeneration:+current+status.">Scaffolds in vascular regeneration: current status.</a> Vasc Health Risk Manag. 11, 79-91 (2015).</li><li>Pektok, 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=Degradation+and+healing+characteristics+of+small-diameter+poly+(e-caprolactone)+vascular+grafts+in+the+rat+systemic+arterial+circulation.">Degradation and healing characteristics of small-diameter poly (e-caprolactone) vascular grafts in the rat systemic arterial circulation.</a> Circulation. 118 (24), 2563-2570 (2008).</li><li>Innocente, F., 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=Paclitaxel-eluting+biodegradable+synthetic+vascular+prostheses:+a+step+towards+reduction+of+neointima+formation?.">Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?.</a> Circulation. 120 (11 Suppl), S37-S45 (2009).</li><li>de Valence, S., 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=Advantages+of+bilayered+vascular+grafts+for+surgical+applicability+and+tissue+regeneration.">Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration.</a> Acta Biomater. 8 (11), 3914-3920 (2012).</li><li>Assmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+autologous+in+vivo+recellularization+of+decellularized+aortic+conduits+by+fibronectin+surface+coating.">Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating.</a> Biomaterials. 34 (25), 6015-6026 (2013).</li><li>Hasan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+scaffolds+for+tissue+engineering+of+vascular+grafts.">Electrospun scaffolds for tissue engineering of vascular grafts.</a> Acta Biomater. 10 (1), 11-25 (2014).</li><li>Baker, B. 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=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29 (15), 2348-2358 (2008).</li><li>Wang, 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=Creation+of+macropores+in+electrospun+silk+fibroin+scaffolds+using+sacrificial+PEO-microparticles+to+enhance+cellular+infiltration.">Creation of macropores in electrospun silk fibroin scaffolds using sacrificial PEO-microparticles to enhance cellular infiltration.</a> Journal of Biomedical Materials Research Part A. 101 (12), 3474-3481 (2013).</li><li>Lee, B. L. P., 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=Femtosecond+laser+ablation+enhances+cell+infiltration+into+three-dimensional+electrospun+scaffolds.">Femtosecond laser ablation enhances cell infiltration into three-dimensional electrospun scaffolds.</a> Acta Biomaterialia. 8 (7), 2648-2658 (2012).</li><li>Rnjak-Kovacina, J., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+pore+size+of+electrospun+scaffolds.">Increasing the pore size of electrospun scaffolds.</a> Tissue Eng Part B Rev. 17 (5), 365-372 (2011).</li><li>Zhong, S., Zhang, Y., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+large+pores+in+electrospun+nanofibrous+scaffolds+for+cellular+infiltration:+a+review.">Fabrication of large pores in electrospun nanofibrous scaffolds for cellular infiltration: a review.</a> Tissue Eng Part B Rev. 18 (2), 77-87 (2012).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+poly(epsilon-caprolactone)+microfiber+and+multilayer+nanofiber/microfiber+scaffolds:+characterization+of+scaffolds+and+measurement+of+cellular+infiltration.">Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.</a> Biomacromolecules. 7 (10), 2796-2805 (2006).</li><li>Rnjak-Kovacina, 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=Tailoring+the+porosity+and+pore+size+of+electrospun+synthetic+human+elastin+scaffolds+for+dermal+tissue+engineering.">Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering.</a> Biomaterials. 32 (28), 6729-6736 (2011).</li><li>Wang, Z., 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+effect+of+thick+fibers+and+large+pores+of+electrospun+poly(epsilon-caprolactone)+vascular+grafts+on+macrophage+polarization+and+arterial+regeneration.">The effect of thick fibers and large pores of electrospun poly(epsilon-caprolactone) vascular grafts on macrophage polarization and arterial regeneration.</a> Biomaterials. 35 (22), 5700-5710 (2014).</li><li>Hutcheson, J. D., 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=Genesis+and+growth+of+extracellular-vesicle-derived+microcalcification+in+atherosclerotic+plaques.">Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques.</a> Nat Mater. 15 (3), 335-343 (2016).</li><li>Tara, S., 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=Well-organized+neointima+of+large-pore+poly(L-lactic+acid)+vascular+graft+coated+with+poly(L-lactic-co-epsilon-caprolactone)+prevents+calcific+deposition+compared+to+small-pore+electrospun+poly(L-lactic+acid)+graft+in+a+mouse+aortic+implantation+model.">Well-organized neointima of large-pore poly(L-lactic acid) vascular graft coated with poly(L-lactic-co-epsilon-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(L-lactic acid) graft in a mouse aortic implantation model.</a> Atherosclerosis. 237 (2), 684-691 (2014).</li></ol>

著者は金融利害の対立があります。

再生の重要な細胞浸潤は、生体内で血管16の改造。限られた細胞浸潤は頻繁に細胞の血管壁への移行を妨げるグラフトの比較的小さな毛穴に関連します。この困難に対処するため大規模な細孔構造を持つエレクトロスピニング PCL 血管を準備する修正法を開発しました。詳しくは、細孔径は処理のパラメーターによって容易に制御できる線維厚の増加に伴い増加しました。結果、宿主細胞が簡単に体内注入後この多孔体血管の壁に浸透し、手術後 12 ヶ月間で明白なセル回帰せず比較的高いレベルで残っている、セル。
ネイティブ動脈主に成っている 3 つの層、内皮、, 中膜と外膜。抗血栓性インターフェイスとして内皮血管の長期開存性を維持する上で重要な役割を果たしています。本研究では、3 ヶ月で移植における血管内皮形成が観察されました。さらに、平滑筋細胞のいくつかの層から成るチュニカ メディアは、血管機能を調節し、動脈の適切な機械的性質を提供することで非常に重要。本研究では、厚い繊維、エレクトロスピニング PCL 移植し、機能チュニカ メディアの再生を著しく強化された大細孔径を明らかにしました。また、再生の平滑筋の構造はネイティブ チュニカ メディアに似ています。平滑筋細胞の収縮の表現型を反映して円周エラスチン ネットワーク内で分散 MYH+細胞のいくつかの層を示した免疫蛍光染色します。重要なは、そのまま組織 (内皮細胞と平滑筋の両方) を再生成し、ECM の合成と分解の不均衡のための 12 ヶ月後も不利な改造はありませんでした。
石灰化は、血管を中心に心血管のインプラントに関連付けられている主要な問題です。血管平滑筋細胞 (VSMCs) は彼らの元の表現を失い方向 osteochondrogenic 血管石灰化のプロセス中に異所性石灰化につながるトランス分化を経験します。注入の 12 ヶ月後も血管壁内で発生するカルシウムの沈着があったことが分かった。マクロ多孔性血管抑制の石灰化の主な理由があります: (1) マクロ多孔性移植片の構造細胞と血液との間のイオン交換などの代謝を促進します。(グラフト構造の 2) の物理的な手がかりが規制または、細胞の1骨芽細胞への分化を抑制する、大きな細孔での (3) の良い細胞浸潤 ECM の分泌を促進、トリガーされますその劣化を抑制します。石灰化17、および (4) の通常または機能 VSMCs カルシウム沈着18を防止する可能性があります。
要約すると、ラット腹部大動脈モデルにおけるマクロ多孔質エレクトロスピニング PCL 血管移植の長期的な評価は、下記の調査を直接分解性血管の潜在的な課題の重要な洞察を提供します。

合成ポリマーから作られた血管は、心血管疾患 (スッテパー) の治療のためのクリニック広く使用されています。残念ながら、小口径人工血管の場合 (D < 6 mm) は、成功した製品が、しばしば血栓症、内膜増殖症などにつながる減らされた血流速によって引き起こされる低開存のために利用合併症1。
ティッシュ ・ エンジニア リングは、長期的な開存性と血管再生の足場誘導と再構成に基づく恒常性を実現するために別の方法を提供します。詳しくは、血管移植三次元テンプレートとしてことができるメカニカル ・ サポートやガイダンスを提供構造維管束組織と影響の細胞機能、細胞接着、移行、増殖などの再生時に、細胞外マトリックス2の分泌。今までは、維管束組織工学用各種合成樹脂を評価されています。これらの高分子の中で poly(ε-caprolactone) (PCL) は良いセルの互換性と数ヶ月から 2 年間3に至るまで劣化を遅らせるために集中的に検討されている.優れた構造健全性開存、増加だけでなく、継続的に細胞浸潤と血管新生ラット大動脈モデル4,5,6、PCL 血管静電紡糸法による処理が展示されて、最大 6 ヶ月の壁を移植します。しかし、細胞と毛細血管と石灰化、回帰を含む不利な組織の改造もで観測した長い縦長を 18 ヶ月。
Cellularization 血管移植の組織再生を決定し、修繕7キー要因であります。エレクトロスピニング、汎用性の高い手法として採用されている広くナノ繊維構造8血管移植の準備のため。残念なことに、比較的小さな細孔構造はしばしばその後の組織再生を制限エレクトロスピニング血管で不十分な細胞浸潤に します。この問題を解決するには、様々 なテクニックは、細孔径と塩/高分子溶出9,10、コレクター装置、レーザー放射11 による治療後の変更を含む、全体的な気孔率を高めるため試行されています。、等。実際には、エレクトロスピニング移植 (ファイバー径、孔径、気孔率を含む) の構造は処理条件12,13に密着します。エレクトロスピニング、中に繊維の直径は、高分子溶液、流量、電圧などの濃度など、パラメーターを変更することにより容易に制御できます。14,15としたがって、細孔径と気孔率がそれに応じて強化され。
我々 は最近、多孔体構造 (5-7 μ m の直径 30-40 μ m の細孔の繊維) と変更された PCL エレクトロスピニング移植を報告しました。ラット腹部大動脈を置き換えることによって体内注入は、3 ヶ月手術後16で開存、として良好な血管内皮形成と平滑筋再生率が高いを示した。もっと重要なは、不利な組織の石灰化とセルの回帰を含む改造が認められなかったのも移植の 1 年後。

<table>
	<tbody>
		<tr>
			<td>Poly(&#949;-caprolactone) (PCL) pellets (Mn=80,000)</td>
			<td>Sigma</td>
			<td>704067</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>A1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Tianjin Fengchuan Company</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley rats</td>
			<td>Laboratory Animal Center of the Academy of Military Medical Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Hebei Tiancheng Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Tianjin Ruijinte chemical company</td>
			<td>2223</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium Injection</td>
			<td>Tianjin Biochem Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin Sulfate Injection</td>
			<td>Jiangsu Lianshui Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-SMA primary antibody</td>
			<td>Abcam</td>
			<td>ab7817</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-smooth MYH primary antibody</td>
			<td>Abcam</td>
			<td>ab683</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-rat elastin antibody</td>
			<td>Abcam</td>
			<td>ab23748</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-von Willebrand factor primary antibody</td>
			<td>Abcam</td>
			<td>ab6994</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150117</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>5% normal goat serum</td>
			<td>Zhongshan Golden bridge</td>
			<td>ZLI9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin (H&#38;E)</td>
			<td>Beijing leagene biotech</td>
			<td>DH0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome</td>
			<td>Beijing leagene biotech</td>
			<td>DC0032</td>
			<td></td>
		</tr>
		<tr>
			<td>Verhoeff-van Gieson (VVG)</td>
			<td>Beijing leagene biotech</td>
			<td>DC0059</td>
			<td></td>
		</tr>
		<tr>
			<td>Von Kossa</td>
			<td>Beijing leagene biotech</td>
			<td>DS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,3-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>G3002b</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,9-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>H901</td>
			<td></td>
		</tr>
	</tbody>
</table>

implantation of electrospun vascular grafts with optimized structure in a rat model

ここでは、マクロポーラス PCL 人工血管を作製し、腹部大動脈置換のモデルラットを用いた評価プロトコルを記述するプロトコルを提案する.エレクトロスピニング血管は頻繁、グラフトに細胞浸潤を制限し、再生とネオ動脈の改造を妨げる比較的小さな毛穴を所有しています。本研究では厚い繊維 (5-6 μ m) と孔径が大きい (~ 30 μ m) PCL 血管を修正処理テクニックを使用して作製しました。腹部大動脈ラットモデルにおける注入によるグラフトの長期的なパフォーマンスを行った。超音波は、グラフト残った動脈瘤や狭窄注入の 12 ヶ月後も発生せず特許を示した。多孔体構造はセル成長を改善し、こうして推進組織 3 カ月で再生成されます。さらに、12 ヶ月後の血管壁内石灰化などの不利な改造の兆候だった。したがって、エレクトロスピニング PCL 血管処理変更した多孔体で保持長期的な注入のため動脈代替可能性を秘めて。

PCL 移植手術後 3ヶ月と 12 ヶ月で仔だったし、ヒアリン マッソン、その行動・ ヴァン ・維 (VVG)、コッサ、蛍光染色法による α-SMA のヘマトキシリンとエオシン (H & E) の標準的な組織学的手法による分析MYH、vWF とエラスチン。組織学的イメージは、正立型顕微鏡を使用して撮影された、ﬂuorescence 顕微鏡を用いた蛍光抗体法により画像が撮影されました。
すべてのデータは、平均 ± SD. 表現されました。2 尾対学生のt-テストの違いを比較に使用されました。Pの値 < 0.05 が統計的に重要であると考えられていた。
新規エレクトロスピニング PCL 移植最適化構造、厚い繊維と孔径が大きいと正常に、本研究で作製しました。平均繊維径が従来より変更された移植 (図 1 a) でほぼ 8 倍厚い SEM 像を示した 1 つ (5.59 ± 0.67 0.69 ± 0.54 μ m 対) (図 1 b)。その結果、平均細孔径が著しく増加から 〜 薄く繊維移植 4.66 μ m 〜 40.88 μ m 厚い繊維 1 つ。断面は、厚い繊維 (図 1-F) と (図 1-私) 薄く繊維グループの両方で鋼管の移植の壁内均一なファイバー分布を示した。壁の厚さは約 400-500 μ m。移植片の機械的性質は、引張試験、特徴づけられた、典型的な応力-ひずみ曲線は図 1に示した。2 移植の機械的性質は伸長の面で明らかに異なっていた。厚い繊維の移植後の対応する値は強化の靭性を示唆、薄く繊維移植に比べて約 3 倍だった。
準備の血管 (内径 2.0 mm)、長さ 1 cm (図 2 a) ラット (図 2 b) ネイティブの腹部大動脈の線分を置き換えるに移植されました。あらかじめ決められた時点で超音波によって移植グラフトの開存性を調べた。結果、移植のほとんどの特許を示した (図 2)。さらに、血流の速度は 12 ヶ月で移植と隣接するネイティブ血管と同様だった。Explanted 移植が動脈瘤 (図 2 D) なしの良い形態を保持し、狭窄や血栓が認められなかったの内腔の表面 (図 2 e)。
組織の再生と 3 ヶ月で ECM 分泌さらに組織解析によって評価されました。H & E 染色 (図 3-H) の血管の内腔に新組織の層が形成されたことを示した。また、vWF の染色を示す内腔が新たに形成された内皮細胞 (図 3 a)、ネイティブの大動脈 (図 3 b) に似たによって完全に覆われていた。一方 MYH 陽性細胞のいくつかの層を示す血管メディア (図 3-D) の再生、円周方向に沿って組織されました。細胞外マトリックスの合成は、マッソンと染色、それぞれ VVG で観測されました。コラーゲンとエラスチン線維性のかなりの量は、血管再生と改造で重要な役割を果たしている移植 (図 3 i-J K-L) 内で観察できます。エラスチンの構造は円周パターンでネイティブの動脈 (図 3E-F) そのような配置は示したさらに蛍光染色します。
さらに、血管内皮細胞と平滑筋の維持を含む再生の組織は統合され、注入 (図 4 a-C) の 12 ヶ月後に逆行でした。もっと重大に、コッサ染色 (図 4) に基づく血管壁内で発生する石灰化の兆候だった。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57340/57340fig1.jpg"> 図 1: 構造および PCL 移植片の機械的特性。厚い繊維 (A) と (B) 繊維が薄いマット PCL はエレクトロスピニングの SEM 画像。厚い繊維管状移植 (D-F)、(G-私) 薄く繊維移植の断面。(C) は、代表的なひずみ-応力曲線を示しています。これらの数字は、趙、らから変更されています。16<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57340/57340fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57340/57340fig2.jpg"> 図 2: 腹部大動脈ラットモデルにおける血管の移植後の着床します。(A) 長さ 1 cm の人工血管をエレクトロスピニング PCL はラット (B) 腹部の大動脈に外科的介在だった。超音波画像は、グラフトが 1 年 (C) 特許生体内であったことを示した。移植された脳動脈瘤 (D) せず隣接するネイティブ大動脈とうまく統合内腔表面がきれい、血栓 (E) の自由表面画像を示します。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57340/57340fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57340/57340fig3.jpg"> 図 3: 組織の再生およびネイティブ大動脈と比較して 3 か月 explanted 移植の ECM の沈着します。再生移植 (A C、およびE) とネイティブの動脈 (B D、およびF) の断面像は、血管内皮細胞、平滑筋細胞やエラスチンを検出する immunostained をだった。H & E 染色ネイティブ大動脈 (H) に比べて explanted 移植 (G) 組織再生を示しています。マッソンの染色 (私) explanted 移植およびネイティブ大動脈 (J) コラーゲンの存在。VVG 染色 explanted 移植 (K) とネイティブ大動脈 (L) のエラスチンの存在を示した。これらの数字は、趙、らから変更されています。16<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57340/57340fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57340/57340fig4.jpg"> 図 4: 12 ヶ月で explanted 移植片の組織学的解析.(A) H & E 染色示した explanted 移植組織の再生。(B) 血管内皮細胞は、vWF 抗体による immunostained だった。(C) 平滑筋だった α SMA 抗体による immunostained です。(D) 石灰化は、コッサ染色で評価しました。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57340/57340fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

エレクトロスピニング ラットモデルにおける最適化された構造を有する人工血管の移植

PCL 血管太い繊維と、大きな細孔を製造するため腹部大動脈置換のラットモデルにおける生体内でのパフォーマンスを評価するためのプロトコルを記述する変更されたエレクトロスピニング法を紹介します。

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

implantation-electrospun-vascular-grafts-with-optimized-structure-rat

Nanyang Technological University

Research

JoVE Journal

Biology

7.9K ビュー.  Nankai University. PCL 血管太い繊維と、大きな細孔を製造するため腹部大動脈置換のラットモデルにおける生体内でのパフォーマンスを評価するためのプロトコルを記述する変更されたエレクトロスピニング法を紹介します。

ビデオ: Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

ラットに到達熟練前肢の構造：運動評価尺度

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

生物学

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

ラット心臓における培養組織の移植

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

胚肢の皮膚の血管系のためのホールマウント免疫組織化学的解析：胚における血管分岐形態形成を研究するモデルシステム

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

マイコプラズマの最適化されたPCRベースの検出

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

マイル アッセイで皮膚の血管の血管誘導剤の評価

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

スクラッチのアッセイの in Vitro細胞遊走の自動光学カメラのテストのために最適化されました。

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

ヒト胚形成期における栄養芽細胞の単一細胞回収

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多パラメータ灌流制御による原発性ラット肝細胞の単離

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

エレクトロスピニング ラットモデルにおける最適化された構造を有する人工血管の移植

エレクトロスピニングラットモデルにおける最適化された構造を有する人工血管の移植