Name: generation and grafting of tissue-engineered vessels in a mouse model
Uploaded: 2015-03-18T14:00:00
Description: Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

This work was supported by The British Heart Foundation and The Oak Foundation.

実験動物の使用とケアのための制度委員会によって承認されたプロトコルに従って、すべての動物実験を行う。 文化メディアの調製<ol><li> F-12K培地、10％ウシ胎児血清（FBS）および100 U / mlペニシリンおよびストレプトマイシン：ヒト線維芽細胞株CCL-153のための培地を加える。 </li><li> PiPSC生成のためのリプログラミングメディアを行いますノックアウトダルベッコ改変イーグル培地（DM...

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現在のプロトコルは、サウンド、機能的な組織工学血管がヒト線維芽細胞からPiPSCを使用して生成することができる、高速で簡単、効率的かつ再現可能な戦略を示している。この技術は、近い将来、再生医療、組織工学および潜在的に患者特異的細胞治療のための貴重なツールを表す。プロトコルの有効性を確保するための重要なステップは、PiPSCの準備、無菌であり、完全に脱細胞化大動脈...

血管導管の建設は、心血管疾患に起因する破損して負傷した血管の外科的修復のための基本的な戦略である。 、同種移植片、自家組織（心膜または伏在静脈）と移植片1、今日まで、外科手術で使用される移植片材料は、生体適合性の合成ポリマー（ポリエチレンテレフタレート[ダクロン】ポ ​​リテトラフルオロエチレン[テフロン（登録商標）]、延伸ポリテトラフルオロエチレン[ゴアテックスのePTFE]）を含む。人工移植片（ 例えば、ゴアテックスおよびダクロン）が最も一般的に使用されている一方で、これらの材料は、おそらく狭窄症、カルシウム沈着、血栓、塞栓および感染を含む多数の短期および長期の合併症を引き起こす。本生物学的な移植片を有する患者は血栓塞栓事象を減少したが、彼 ​​らはまだ石灰化の劣化2のためにこのような二次移植の失敗と短縮耐久性などの制限が発生した。したがって、外科トンの大幅な改善にもかかわらず年間のechniquesは、研究者や臨床医は、まだ血管疾患のための理想的なコンジットを識別するための必要性を抱えている。さらに最近では、血管組織工学の研究分野は、細胞が正常な移植の1のための機能的血管を具現化するバイオミメティック環境を作ることを目的に、生分解性足場に組み込まれている概念を生成した。基本的に、血管の構築体の成功は3必須成分に依存。足場を含む細胞、 すなわち、内皮細胞の内層と、平滑筋細胞層、天然の血管系に匹敵する機械的特性を提供するために適切な細胞外マトリックスを含有する足場、および開始/調節するために必要とされる分子/細胞シグナル修理。 長期グラフト開存とネオ·組織の持続的な発展は、番目の足場の効果的な細胞播種に大きく依存しているereby決定的な重要性の細胞型の決定をレンダリングする。いくつかの報告は、小径導管3-6を開発するためにさまざまなソースから成熟内皮および平滑筋細胞の使用を実証する。有望であるが、成熟した内皮細胞および平滑筋細胞を得るのに十分な自家血管の欠如は、大きな負担残る。より最近では、様々な供給源からの幹細胞は、血管組織工学用途のために開発されてきた。実際には、胚性幹細胞7を含む幹細胞型の多様な、誘導多能性幹細胞（iPS細胞）8,9、PiPSC 10,11、骨髄由来単核球12、間葉系幹細胞13、内皮前駆細胞および成体血管壁14,15は 、定義されたすべてのメディアに対応して機能的な内皮細胞または平滑筋細胞のいずれかに分化できることが実証されている細胞抗原-1（のSca-1）+幹/前駆細胞を幹細胞由来と培養条件。さらに、幹細胞の無制限の自己再生能力が彼らにのみ増殖停止および老化を受ける前に有限回数に分割することができる成熟内皮および平滑筋細胞とは異なり、より良い候補者を作る。 移植のために成功した組織工学血管を生成するための足場材料の選択は、生体適合性、生体力学的特性、および生分解速度のようないくつかの要因に依存する。基本的には、移植片のための足場を作成するために使用される材料は、生分解性であるべきであり、不必要な受信者の免疫応答をマウントしません。加えて、細胞付着およびその後の生存に適し多孔性およびミクロ組織を包含する必要があります。現在までに、血管組織工学における足場のために使用される最も一般的な材料は、ポリグリコール酸、ポリ乳酸、ポリεカプロラクトン16のポリマーが挙げられる。さらに最近では、脱細胞化生物学的材料が持っているまた、ある程度の成功を収めて適用されて。いくつかの研究室は、自己細胞で脱細胞化ヒト、イヌ又はブタ血管を播種すると、凝固し、内膜肥厚17-19抵抗し、生物学的移植片を提供したことが示されている。血管組織工学における他の戦略は、フィブリンゲル13中に細胞を播種し、足場支持体20、21せずに細胞シートを生成し、例えば 、細胞外マトリックスタンパク質ベースの血管移植片が含まれる。 現在のプロトコルは、SCID（重症複合免疫不全に官能内皮細胞および平滑筋細胞、機能的なPiPSC由来の血管細胞を港に脱細胞化血管足場から成るバイオリアクターの生成、および組織工学血管のグラフトへのヒトPiPSCの分化を実証）マウス。 PiPSCは、これらの細胞がマウスにおいて腫瘍を形成するか、または倫理的な発生させないので、血管移植片の組織工学に使用する最適な細胞型であると同種免疫応答。さらに、我々は、PIPS内皮細胞およびPIPS平滑筋細胞を生成するための戦略は、10,11、効率的かつ再現可能であることを示している。その後、我々はこのようにグラフトと生存効果を増強する、密接にネイティブ血管内に存在するマトリックスタンパク質を模倣するPiPSC由来の血管細胞の播種のための脱細胞化容器を設計しました。さらに、従来PiPSC播種に血管の脱細胞はマクロファージなどの免疫細胞の種類によってマウント炎症反応の発生を防止する。さらに重要なことに、このプロトコルは、ヒトへの翻訳のための有望な血管の導管を生成する方法を表すだけでなく、マウスモデルを介して、血管組織再生を支配する分子メカニズムを研究し、理解する貴重な手段を提供しないだけでなく。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Human Fibroblasts CCL-153</td>
			<td >ATCC</td>
			<td >CCL-153</td>
			<td>Prenatal human embryonic fibroblasts</td>
		</tr>
		<tr >
			<td >ATCC F-12K Medium (Kaighn&#39;s Modification of Ham&#39;s F-12 Medium)</td>
			<td >ATCC</td>
			<td >30-2004</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ATCC</td>
			<td >30-2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout DMEM medium optimized for embryonic stem cells</td>
			<td >Life technologies (Gibco)</td>
			<td >12660-012</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout Serum Replacement</td>
			<td >Life technologies (Invitrogen)</td>
			<td >10828-028</td>
			<td></td>
		</tr>
		<tr >
			<td >Human Basic FGF-2&#160;</td>
			<td >Miltenyi Biotech</td>
			<td >130-093-837</td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Life technologies (Invitrogen)</td>
			<td >32571093</td>
			<td></td>
		</tr>
		<tr >
			<td >Human PDGF</td>
			<td >R&#38;D System</td>
			<td >120-HD-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Gelatin Solution 2%</td>
			<td >Sigma</td>
			<td >G1393</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td>
			<td >Addgene</td>
			<td >20866</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;PvuI Restriction Enzyme</td>
			<td >New England Biolabs</td>
			<td >RO150S</td>
			<td></td>
		</tr>
		<tr >
			<td >SureClean Plus</td>
			<td >Bioline</td>
			<td >BIO-37047</td>
			<td></td>
		</tr>
		<tr >
			<td >Nucelofection Kit (NHDF Kit)</td>
			<td >LONZA</td>
			<td >VPD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Neomycin</td>
			<td >SIGMA</td>
			<td >G418</td>
			<td>Selection of&#160;</td>
		</tr>
		<tr >
			<td >KL 1500 LCD, Illumination for Stereo Microscopy</td>
			<td >SCHOTT</td>
			<td >KL 1500 LCD</td>
			<td>Cold light illumination for stereo microscopy</td>
		</tr>
		<tr >
			<td >Nikon Zoom Steromicroscope SMZ800</td>
			<td >Nikon</td>
			<td >SMZ800</td>
			<td></td>
		</tr>
		<tr >
			<td >Heparin sodium salt</td>
			<td >Sigma</td>
			<td >H3393</td>
			<td></td>
		</tr>
		<tr >
			<td >10% SDS Stock Solution Molecular Biology Reagent</td>
			<td >Severn Biotech</td>
			<td >CAS 151-21-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td >Sigma</td>
			<td >D8537</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrigel (10mg/ml)</td>
			<td >BD</td>
			<td >A6661</td>
			<td></td>
		</tr>
		<tr >
			<td >Shaker IKA Vibrax with Shaking platform VX 7&#160;</td>
			<td >Jepson Bolton&#39;s, Janke&#38;Kunkel</td>
			<td >S32-102</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S Digital Pump Drive</td>
			<td >Cole-Parmer</td>
			<td >WZ-07523-80</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S 6-channel, 6-roller cartridge pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S large cartridges for pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-75</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td>
			<td >Cole-Parmer&#160;</td>
			<td >WZ-96410-14</td>
			<td >Tubing goes through the peristaltic pump</td>
		</tr>
		<tr >
			<td >0.5mm ID, 0.8 mm OD Silicone Tubing</td>
			<td >SILEX</td>
			<td >N/A</td>
			<td>Tubings connect incubation chamber, media reservoir and compliance chamber&#160;</td>
		</tr>
		<tr >
			<td >Fitting Reducer 0.5 to 1.6, natural Polypropyline</td>
			<td >Ibidi</td>
			<td >10829</td>
			<td>Adapter connect above two types of tubings</td>
		</tr>
		<tr >
			<td >1/32&#34; Tubing, ID 0.01&#34; (250&#181;m) Material: PEEK</td>
			<td >LabSmith</td>
			<td >T-132-010P</td>
			<td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td>
		</tr>
		<tr >
			<td >One-Piece Fittings&#160;</td>
			<td >LabSmith</td>
			<td >T-132-100</td>
			<td>Fix the above tubings through the incubation chamber wall</td>
		</tr>
		<tr >
			<td >Nylon tubes (OD 0.9mm, ID 0.75mm)&#160;</td>
			<td >Smiths Medical</td>
			<td >N/A</td>
			<td>Tubings insert into two ends of the aorta graft</td>
		</tr>
		<tr >
			<td >NOD.CB17-Prkdcscid/NcrCrl mouse</td>
			<td >Charles River</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 8-0&#160; silk</td>
			<td >ETHICON</td>
			<td >W819</td>
			<td ></td>
		</tr>
		<tr >
			<td >Hypnorm</td>
			<td >Vetapharm</td>
			<td >Vm21757/4000</td>
			<td >Neuroleptanalgesic for use in mice</td>
		</tr>
		<tr >
			<td >Hypnovel (Midazolam)</td>
			<td >Roche</td>
			<td >59467-70-8</td>
			<td >Induction of anaesthesia</td>
		</tr>
		<tr >
			<td >Dissecting microscope</td>
			<td >Carl Zeiss</td>
			<td >Stemi 2000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nylon Tubing</td>
			<td >Portex LTD</td>
			<td >800/200/100/200</td>
			<td >0.65 mm in diameter and 1 mm in length; to make artery cuff</td>
		</tr>
		<tr >
			<td >Electrocoagulator</td>
			<td >Martin&#160;</td>
			<td >SN 54.131</td>
			<td >Ligation of artery branches on aorta</td>
		</tr>
		<tr >
			<td >Bipolar micro hemostat forceps</td>
			<td >Martin</td>
			<td >80-91-12-04</td>
			<td >Fixation of vessel ends</td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFX-7</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFL-3dZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >D-5aZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini applier&#160;</td>
			<td >AESCULAP</td>
			<td >FE572K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micro hemostats clips</td>
			<td >AESCULAP</td>
			<td >FE720K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 6-0 VICRYL</td>
			<td >ETHICON</td>
			<td >V489</td>
			<td ></td>
		</tr>
	</tbody>
</table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

PiPSCの成功した世代は、4転写因子、OCT4、SOX2、KLF4およびc-MYC（OSKM）を運ぶ線形化さpCAG2LMKOSimOプラスミドでヒト線維芽細胞をnucleofectingた後、4日確認された。 PiPSCは、線維芽細胞（ 図2A）と比較した場合に顕著に異なる表現型を表示し、mRNA（ 図2B）およびタンパク質（ 図2C）レベル10で4の再プログラミング因子を発現した。 PiPSCベースの血管移植...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

ここでは、二重播種部分的人工多能性幹細胞がマウスにグラフトするための機能的な組織工学血管移植片を生成するためのプロトコルを提示する（PiPSC）は - 脱細胞化血管足場バイオリアクターに由来内皮細胞 - 平滑筋細胞およびPiPSC由来。

マウスモデルにおける組織工学血管の生成とグラフト

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Research

JoVE Journal

Bioengineering

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

トライリーフレットポリマーバルブの相対的な流体力学的評価のためのプロトコル

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

複合表面グラフトと後置修飾法による光応答膜の調製

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

組織工学血管移植片とその後の移植のために手術手技<em&gt;インビボ</em&gt;モニタリング

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

操作された複合三次元組織<em&gt;インビトロ</em&gt; /<em&gt;インシリコ</em具体的な突然変異背景に薬物の有効性を予測するための&gt;肺腫瘍モデル

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

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

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

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

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

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

三次元組織配置に発達メカニズムを要約し、神経系の再生を容易にするアストロ サイト ネットワーク

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

大きな腱欠損を修復する組織設計されたコラーゲン移植片を用いた新規テノルラフィー縫合技術

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

シードとマウス モデルにおける合成組織設計気管移植片の移植

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...