著者らは、部分的に研究活動をサポートし、FP7 BiophotonicPlusプロジェクト「LITE」が付与されたトスカーナ地方（POR CREO FESR 2007-2013、坂東ユニコR＆S 2012）から資金提供を受けFORTEプロジェクト、EU FP7 ECHORD ++実験LA-ROSESに感謝したいですトスカーナ地域による。

研究は、病院の倫理委員会の承認を得て、将来に向かって行きました。インフォームドコンセントを得ました。研究は、ヘルシンキ宣言の教義を遵守していました。 1.ドナー内皮準備<ol><li>室温でローカルアイバンクにより調製したドナー角膜を使用してください。 </li><li>手術室では、その送達コンテナからドナー角膜を引き出し、角膜の輸送に使用されている組織保存と栄養のためのソリューションを離れて設定します。 </li><li>液体が流れ、可変圧力を作成することができますその中に人工前房、上のドナー角膜 - 強膜リムを配置。組織保持ヘッドで角膜をカバーしています。 </li><li>角膜の保存と栄養の液体で満たされたシリンジに3ウェイコネクタを介して人工前房（AAC）を接続します。 </li><li>所定の位置にしっかり保持器を保持するために圧縮リングを使用してください。 </li><li> SYRを使用してください（1.7参照）を最適圧力に達するまで組織保存溶液で前房を満たす：房内の圧力を維持するために、AACに接続インゲ。コネクタを閉じます。 </li><li>指で前方のチャンバー内の圧力をテストします。 （範囲60〜70 mmHgの中で）右内の圧力に到達するまでの内部の圧力を変更します。 3ウェイコネクタを閉じます。 </li><li>光コヒーレンスThomography（OCT）とドナー角膜の厚さを測定します。機器の光学系の前に固定された位置に、その角膜と前房を維持した後、全厚-OCT取得を取ります。 </li></ol>ドナー内皮の2フェムト秒レーザーの準備<ol><li>フェムト秒レーザ装置の下でドナー角膜の圧平を実行します。後部サイドカット、完全な層状と前サイドカット：3その後のカット​​で、ドナー組織を切断するfemtosecレーザーを使用してください。 3カットのために、以下のパラメータを設定します。<ol><li>フルラメラカット（ラスターパターン、アウトスタート）の場合は、必ず95ミクロンを減算、ドナー角膜（1.8で説明したように10月で測定）の最も薄い点に対応する切り込み深さを設定します。範囲（作業深さに応じて）0.8〜0.9μJにパルスエネルギーを設定します。 8.7ミリメートルに直径を設定します。 2ミクロンの接線スポット分離を設定します。 2μmのラジアルスポット分離を設定します。 </li><li>前方サイドカットについては、前回の完全ラメラカット30μmより深い後部の深さを設定します。 2.10μJにパルスエネルギーを設定します。 8.6ミリメートルの直径。層分離しながら3以上3μm以下にスポット分離、。 </li><li>後部サイドカットのために、後側の前方深さを見つける前方フルラメラカット30μmよりをカット。 900ミクロンの後方の深さを設定します。 2.10μJにパルスエネルギーを設定します。 8.3ミリメートルの直径。 2ミクロンのスポット分離; 2ミクロンの層分離。 </li></ol></lI&gt; </ol> 3.受信者の目の準備<ol><li>手術のために患者を準備します。刃を予め較正4.0ミリメートルと12時1 4.00ミリメートル角膜切開を行います。 2時位置に配置された1.2ミリメートル予め較正刃と1輪部穿刺を作ります。 30°刺すナイフで6時位置に別の輪部穿刺を行います。 </li><li> 2時穿刺を介して、患者の前室に前室のメンテナを挿入します。 </li><li>専用フックで1 8.2ミリメートルの直径の円形descemetorexisを実行します。 </li><li>後部間質からデスメ膜および内皮を除去し、デスメフックで組織を削除します。 </li></ol> 4.発色団の準備<ol><li> 1.5ミリリットルマイクロチューブにインドシアニングリーン粉末1mgを置きます。 </li><li>滅菌水の9 mgの追加（ICG水溶液はw / wの10％です）。 </li><li>手動金属攪拌機を備えたICG粉末と水を混ぜます。 </lI&gt; </ol>ドナー内皮の5染色<ol><li>インジェクタ表面に接触する内側と、ビジネスサービスインジェクタの広い部分にドナー内皮を置きます。 </li><li>スパチュラを使用して、その周辺部に、発色団溶液でドナー内皮の内側を染色します。正しく染色された組織は、均一な緑がかった色をしています。ステップ5.3を起動する前に3分を待ちます。 </li><li>ビジネスサービスインジェクタの前方部分にドナー内皮を引き出し、ロールバックされます。 </li></ol> 6.ドナー内皮の挿入<ol><li>内皮レンチキュラのための非外傷性の同軸鉗子を使用して、折り畳まれたドナー内皮をつかみ、挿入します。 </li><li>前房のメンテナを削除します。 </li><li>ナイロン10.0単一シュティッヒと角膜切開と（ステップ3.1で実行）穿刺を縫合。 </li><li>ドナー組織を展開し、受信者の角膜に対してそれを押すように気泡を注入します。気泡必見カンプletely前房の空間を埋めます。 </li></ol> 7.レーザー溶接<ol><li>フックやヘラで角膜の外側から気泡を移動させると内側から、受信者の角膜の中心にドナーレンチキュラを配置します。 </li><li>開口数（NA）として0.22で、300μmのコア径の滅菌光ファイバを装備した810 nmの発光ダイオードレーザーを使用します。 </li><li>眼球外ファイバ先端を維持し、透明な角膜組織を通して、染色された内皮に向かってレーザ光を実現します。ファイバチップは、非接触構造です。 </li><li>単一スポット発光モード、70〜80ミリ秒のパルス持続時間、パルス当たり35〜40ミリジュール：レーザーは、次の設定を使用します。彼は-Neが上のビームを目指しています。 </li><li>ドナー内皮の染色周囲に単一のレーザスポットを提供します。順次スポットを配信：最終的な側面は、ドナーレンチキュラの周辺のスポットの環です。 2 ADJA間の距離セントスポットセンタースポット径のダブルです。 </li><li> 0.3％トブラマイシンおよび0.1％デキサメタゾン眼科用懸濁液と一緒に、患者の目にコンタクトレンズを適用します。 </li></ol>

<ol>
	<li>Tan, D. T., Dart, J. K., Holland, E. J., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+transplantation.">Corneal transplantation.</a> Lancet. 379 (9827), 1749-1761 (2012).</li><li>El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmol Ther. , (2014).</li><li>Rossi, 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=Laser+tissue+welding+in+ophthalmic+surgery.">Laser tissue welding in ophthalmic surgery.</a> J Biophotonics. 1 (4), 331-342 (2008).</li><li>Pini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+femtosecond+laser+ablation+and+diode+laser+welding+in+lamellar+and+endothelial+corneal+transplants.">Combining femtosecond laser ablation and diode laser welding in lamellar and endothelial corneal transplants.</a> Proc. SPIE Ophthalmic Technologies XVIII. 6 (844), 684411-1-684411-7 (2008).</li><li>Rossi, 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=All-laser'+endothelial+corneal+transplant+in+human+patients.">All-laser' endothelial corneal transplant in human patients.</a> Proc. SPIE Ophthalmic Technologies XXII. 8209, 82091O-1-82091O-3 (2012).</li><li>Rossi, F., Pini, R., Menabuoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+model+analysis+on+the+temperature+dynamics+during+diode+laser+welding+of+the+cornea.">Experimental and model analysis on the temperature dynamics during diode laser welding of the cornea.</a> J Biomed Opt. 12 (1), 014031-1-014031-7 (2007).</li><li>Menabuoni, 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=Laser-assisted+corneal+welding+in+cataract+surgery:+retrospective+study.">Laser-assisted corneal welding in cataract surgery: retrospective study.</a> J Cataract Refract Surg. 33 (9), 1608-1612 (2007).</li><li>Buzzonetti, 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=Laser+Welding+in+Penetrating+Keratoplasty+and+Cataract+Surgery+in+Pediatric+Patients.+Early+Results.">Laser Welding in Penetrating Keratoplasty and Cataract Surgery in Pediatric Patients. Early Results.</a> J Cataract Refract Surg. 39 (12), 1829-1834 (2013).</li><li>Menabuoni, 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=The+'anvil'+profile+in+femtosecond+laser-assistedpenetrating+keratoplasty.">The 'anvil' profile in femtosecond laser-assistedpenetrating keratoplasty.</a> Acta Ophthalmol. 91 (6), e494-e495 (2013).</li><li>Canovetti, 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=Laser-assisted+penetrating+keratoplasty:+one+year&#8217;s+results+in+patients,+using+a+laser-welded+&#8220;anvil&#8221;-profiled+graft.">Laser-assisted penetrating keratoplasty: one year&#8217;s results in patients, using a laser-welded &#8220;anvil&#8221;-profiled graft.</a> Am J Ophthalmol. 158 (4), 664-670 (2014).</li></ol>

The authors declare that they have no competing financial interests.

「すべてのレーザー「内皮移植は低侵襲性角膜移植に独創的なアプローチです。 プロトコル内で説明するすべての手順は、滅菌手袋、ガウン、マスクとキャップの使用などの手術中に一般的な慣行である衛生と消毒の手順を観察し、手術室で行いました。 ICG溶液は、すぐにドナー内皮の染色におけるその適用前に、手術室で調製しました。 ICG粉末、水および染色溶液?...

本研究では、ドナー組織と受信者のベッドの上に溶接する近赤外ダイオードレーザーを製造するためのフェムト秒レーザーの使用に基づいて内皮角膜移植への独創的なアプローチを提示します。ドナー角膜の術中測定が正しくドナー組織の大きさを設計する必要があります。内皮角膜内皮疾患1,2の治療に貫通角膜移植を置き換えるために提案されています。この技術の主な利点は、手術、移植片拒絶のリスクが減少し、目の整合性の保存時に角膜移植、減少麻酔を貫通に対して、より高速視力回復です。主なリスク要因は、手術後のドナーレンチキュラー転位です。標準的な技術は、それが気泡の注入によって維持され、その最終的な位置にドナー内皮を挿入することによって実行されます。何の縫合糸があるため、機械的な生物物理学的および次元キャラの使用されていません内皮のcteristics。また、視力の回復が原因で、移植厚い組織に主な理由は、ドナーとレシピエントの組織との間のミスマッチのために制限することができます。 ここでは、これらの主要な問題を克服することができる内皮角膜移植を行う際の手順を紹介します。ドナー内皮はレーザー溶接技術を用いて、その最終位置に確保することができます。これは、制御され、ローカライズされた光熱過程である：それは、ドナー/レシピエントの界面に誘導することができます。これは、過去10年間に研究し、角膜を貫通および内皮3-5の移植で提案されています。近赤外光（波長810 nm）の低電力ダイオードレーザーによって放射され、創傷部位での生体組織に向かって送出されます。角膜は、この波長に対して透明であり、当然ながら、レーザ光を吸収するために、この組織を作るためには、発色団でそれを染色する必要があります。提案された染料は、無菌飽和でありますインドシアニングリーン（ICG）のd水溶液。我々は、角膜組織が ​​適切にこのICG製剤で染色されている場合、それは810ナノメートル6に吸収ピークを示すことを実証しました。また、ICGは、広く臨床診断で使用され、その安全性は既にヒト被験体において実証されています。染色された角膜は、ダイオードレーザー光のエネルギーを吸収し、得られた主な効果は、溶接部位での制御された温度上昇です。熱的効果は無染色の組織において誘導されていません。温度拡張機能は、冷却時の傷の壁のすぐclosuringで、間質コラーゲンで可逆的熱変性を誘導します。このレーザ溶接効果がまず白内障手術7,8及び角膜移植9,10を貫通するに実証されました。私たちはこの論文で提示されている最適化されたアプローチは、内皮角膜移植に応用するために研究されています。 提案された手術では、単一のレーザスポット（ミリ秒の持続十）AReは、スポット寸法（直径ミクロンの数百）内に局在光熱効果で、その結果、組織に送達：誘導された効果は、ドナー/ホストインターフェイスに閉じ込められたコラーゲンの光凝固からなる、ハードレーザー溶接です。溶接部位でのコラーゲンの変性の結果は、このように標準的な技術（ステッチ）で取得することは不可能である縫合効果を提供する、ドナーとホスト組織との間に強い接着力です。組織は、短いフォローアップ（1ヶ月）に彼の自然な外観を取り戻し、ドナー/宿主組織との間の接着は、治癒期の非常に早い段階で提供溶接することによって改善されます。 厚いドナー組織の移植、すなわち内皮角膜移植の他の主要な危険を回避するために、intrasurgical光コヒーレンストモグラフィー（OCT）を使用する：正しいカットプロファイルで設計することができるように、商用デバイスは、ドナー角膜の厚さを測定しますザfemtosecレーザー。提案された「すべてのレーザー「内皮移植は、このように、この低侵襲手術の臨床結果を改善するように思われます。

<table><tbody><tr><td>Indocyanine Green</td><td>Pulsion Medical Systems, Germany</td><td>ICG-PULSION (http://www.pulsion.com/international-english/perfusion/icg-pulsion/)</td><td>Alternative product: IC-GREEN, Akorn&amp;nbsp; Inc., Lake Forest, Illinois- US (http://www.icginjection.com/)</td></tr><tr><td>Femtosecond Laser&amp;nbsp;</td><td>Abbott Medical Optics,&amp;nbsp; Abbott Laboratories Inc. Abbott Park, Illinois, USA</td><td>iFS150 (http://www.abbottmedicaloptics.com/products/refractive/ilasik/ifs-advanced-femtosecond-laser)</td><td></td></tr><tr><td>Optical Coherence Tomography (OCT)</td><td>Carl-Zeiss Meditec, Dublin, California- US (http://www.zeiss.com/meditec/en_de/home.html)</td><td>Visante</td><td></td></tr><tr><td>Diode Laser</td><td>E.l.En. Group s.pa., Calenzano-FI, Italy (http://www.elengroup.com/en/divisions/medical)</td><td>Mod. WELD 800</td><td></td></tr><tr><td>Artificial Anterior Chamber</td><td>CORONET, corneal graft products. Network Medical Products Ltd. Coronet House, Kearsley Road, Ripon, North Yorkshire, HG4 2SG, UK</td><td>Artificial Anterior Chamber (A.A.C.) with large and small tissue-retaining heads. Code 51-935 (http://www.networkmedical.co.uk/ophthalmic_artificial_&lt;br /&gt;anterior_chamber.html)</td><td></td></tr><tr><td>Solution for tissue preservation and nutrition&amp;nbsp;&amp;nbsp;</td><td>AL.CHI.MI.A. Srl, Viale Austria 14, 35020 - Ponte S. Nicol&amp;ograve; - PD&lt;br/&gt; &amp;nbsp;ITALY</td><td>Carry-C media for corneal deturgescence and transport at room temperature - 12 x 50 ml (http://www.alchimiasrl.com/en/organ-culture-at-31&amp;deg;-c-eb/carry-c-eb)</td><td></td></tr></tbody></table>

an &#8220;all-laser&#8221; endothelial transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

「全てのレーザ」外科手術を低侵襲角膜移植を行うことが提案されています。手順は、（ 図1参照）を実行するのは簡単である：標準内皮移植に関して、角膜の厚さを測定ドナー組織を染色し、レーザー光を送達する唯一のステップが追加されます。達成される利点は、主に、数分の増加の外科時間を補償します。ドナー角膜の厚さとドナーレンチキュラー寸法をカスタマイズ?...

Watch this Scientific Journal Video about An Ã¢â‚¬Å“All-laserÃ¢â‚¬Â Endothelial Transplant at JoVE.com

An &#8220;All-laser&#8221; Endothelial Transplant

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

「オールレーザー」内皮移植

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Research

JoVE Journal

Medicine

9.4K ビュー.  Italian National Research Council. An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

ビデオ: An “All-laser” Endothelial Transplant

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

大動物モデルにおける心血管デバイス用自家血管内皮前駆細胞播種技術と生体適合性試験

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

生物工学

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

内皮角膜移植のために角膜ドナー組織の準備

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

医学

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

後肢虚血の治療のための胚性幹細胞由来血管内皮細胞

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

生物学

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

縦用の眼の前房に移植、非侵襲<emインビボ&gt;</emリアルタイムでのシングルセル分解能&gt;イメージング

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet&#39;s membrane) is introduced onto the recipient&#39;s posterior stroma3. Unlike Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity4. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision1. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet&#39;s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK5. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet&#39;s membranes from donor corneoscleral buttons for transplantation2.

デスメ膜内皮角膜移植のための角膜ドナー組織の準備

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

設計の内皮細胞とひと卵巣組織の共同移植: セル ベースの戦略の組み合わせは直接パラクリン配信による血液灌流を加速

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮細胞損失の非侵襲的レーザー支援実験モデルの開発

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

操作された線維芽細胞増殖因子によって刺激された加速治癒を伴うデスメのストリッピングのみのヒト角膜器官培養モデル1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

地図状委縮の目の大きいモデルにヒト胚性幹細胞由来網膜色素上皮細胞の網膜下移植

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

内皮から間葉への移行を研究するための生体内心臓移植

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

著者スポットライト:内皮から間葉への移行の研究のための新生児ヘテロトピックラット心臓移植モデル  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

内皮から間葉への移行研究のための新生児異所性ラット心臓移植モデル

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...

「オールレーザー」内皮移植

要約

さらに動画を探す

この動画の章

大動物モデルにおける心血管デバイス用自家血管内皮前駆細胞播種技術と生体適合性試験

内皮角膜移植のために角膜ドナー組織の準備

後肢虚血の治療のための胚性幹細胞由来血管内皮細胞

縦用の眼の前房に移植、非侵襲

デスメ膜内皮角膜移植のための角膜ドナー組織の準備

設計の内皮細胞とひと卵巣組織の共同移植: セルベースの戦略の組み合わせは直接パラクリン配信による血液灌流を加速

角膜内皮細胞損失の非侵襲的レーザー支援実験モデルの開発

操作された線維芽細胞増殖因子によって刺激された加速治癒を伴うデスメのストリッピングのみのヒト角膜器官培養モデル1

地図状委縮の目の大きいモデルにヒト胚性幹細胞由来網膜色素上皮細胞の網膜下移植

内皮から間葉への移行研究のための新生児異所性ラット心臓移植モデル

「オールレーザー」内皮移植

要約

さらに動画を探す

この動画の章

大動物モデルにおける心血管デバイス用自家血管内皮前駆細胞播種技術と生体適合性試験

内皮角膜移植のために角膜ドナー組織の準備

後肢虚血の治療のための胚性幹細胞由来血管内皮細胞

縦用の眼の前房に移植、非侵襲

デスメ膜内皮角膜移植のための角膜ドナー組織の準備

設計の内皮細胞とひと卵巣組織の共同移植: セル ベースの戦略の組み合わせは直接パラクリン配信による血液灌流を加速

角膜内皮細胞損失の非侵襲的レーザー支援実験モデルの開発

操作された線維芽細胞増殖因子によって刺激された加速治癒を伴うデスメのストリッピングのみのヒト角膜器官培養モデル1

地図状委縮の目の大きいモデルにヒト胚性幹細胞由来網膜色素上皮細胞の網膜下移植

内皮から間葉への移行研究のための新生児異所性ラット心臓移植モデル

設計の内皮細胞とひと卵巣組織の共同移植: セルベースの戦略の組み合わせは直接パラクリン配信による血液灌流を加速