Name: in vitro and in vivo models to study corneal endothelial-mesenchymal transition
Uploaded: 2016-08-20T14:00:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

すべての手順は、眼科と視覚研究における動物の使用のためのビジョンと眼科計算書の研究のための協会に従う本研究で続き、国立台湾大学病院の施設内動物管理使用委員会によって承認されました。 1.単離、初代培養の準備、およびウシのCECの免疫染色<ol><li>地元の食肉処理場から新鮮な牛の目を獲得。 </li><li> 3分間10％w / vのポビドンヨード溶液中で目を消毒。リン酸緩衝生理食塩水（PBS）溶液でそれらを洗浄します。 </li><li>無菌条件下でのメスやハサミで角膜ボタンを収穫。ピール解剖顕微鏡下でピンセットでデスメ膜は、（本研究では、ウシ目は地元の食肉処理場により摘出していることに注意してください。したがって、最初の研究手順は研究室でpreenucleated目の消毒ありました）。 </li><li>トライの1ミリリットルでデスメ膜をインキュベート37℃で30分間PSIN。 5分間112×gで遠心分離することによってウシのCECを収集します。補充ホルモン上皮培地（SHEM）HEPES緩衝ダルベッコ改変イーグル培地、5％ウシ胎児血清を補充したハムF12培地、0.5％のジメチルスルホキシド、人間の表皮の2 ng / mlでの等量を含有する1mlの細胞を再懸濁成長因子、インスリンの5 mg / mlで、トランスフェリンの5 mg / mlで、セレン5ngの/ mlのコレラ毒素の1ナノモル/ Lのゲンタマイシンを50mg / mlであり、アンフォテリシンBの1.25 mg / mlで</li><li> 6センチメートル皿に細胞（眼あたり約1×10 5細胞）をシード。 SHEM文化それらを。空気中5％のCO 2の雰囲気中、37℃で皿をインキュベートします。 3日ごとに培地を変更します。 </li><li>細胞がコンフルエントに達したとき、PBSでそれらを洗浄し、5分間37℃でトリプシン1mlにそれらをインキュベートします。 5分間、112×gで遠心分離することによって、それらを収集します。 SHEMの1ミリリットル中に細胞ペレットを再懸濁します。 <ol&gt; <li>血球計で細胞をカウントします。 24ウェルプレート中のウェル当たり1×10 4の密度、および文化SHEM中の細胞でカバースライド上の細胞をシード。マリマスタットのEnMT抑制効果を調査するために、培養培地に添加しマリマスタットの10μMとSHEM中で細胞をインキュベートし、3日ごとに培地を変更します。 </li></ol></li><li>室温で30分間、4％パラホルムアルデヒドを250μl、pH7.4で指示された時点で細胞を固定してください。 5分間、0.5％トリトンX-100250μlの透過性。 30分間、10％のウシ血清アルブミンでブロックします。 </li><li> 4℃で一晩アクティブβ-カテニン（ABC）、カタツムリ、およびナメクジに対する一次抗体で細胞をインキュベートします。用いた一次抗体の希釈は1：200。抗体を10mMのPBS、1％w / vのウシ血清アルブミン、および0.09％のw / vアジ化ナトリウムからなる抗体希釈液で希釈します。 </li><li> 15分間、PBSで2回細胞を洗浄し、インキュベート室温で1時間：アレクサフルオロ結合二次抗体（抗体希釈液で100 1）を持つメートル。 </li><li> 4 &#39;、6-ジアミジノ-2-フェニルインドールを2μg/ mlの細胞をカバーすることにより細胞核対比、15分間、PBSで2回細胞を洗浄し、取り付け溶液抗退色でそれらをマウント。 </li><li> 20X対物倍率でレーザ走査共焦点顕微鏡を用いて405および488nmでの励起波長での免疫蛍光画像を得ます。 </li></ol> 2.ラット角膜内皮Cryoinjuryモデルと房内注入<ol><li> 2％キシラジン（5.6ミリグラム/キログラム）とチレタミンプラスゾラゼパム（18ミリグラム/キログラム）の筋肉内注射で12週齢の雄性Sprague-Dawleyラットを麻酔。優しく肌のけいれんの不在下で適切な麻酔を確認するために、動物の皮膚をつまみます。 </li><li>眼の痛みや瞬目反射を最小限に抑えるために、各ラットの右眼に0.5％塩酸プロパラカインを一滴を植え付けます。植え付けます角膜の乾燥を防ぐために、左眼にテトラサイクリン軟膏。 </li><li>液体窒素中でステンレススチールプローブ（直径= 3 mm）を冷却します。 30秒間、右眼の角膜中央にステンレス製のプローブを適用します。頻繁に角膜の乾燥を防ぐための処置中に右眼にPBSを植え付けます。 </li><li>すぐcryoinjuryた後、0.1％アトロピンおよび0.3％の硫酸ゲンタマイシンを植え付けると、一日一回毛様体のけいれんに起因する眼の痛みを和らげるために、感染を防ぐために。 <ol><li>手順の後、ヒートランプを使用して温め、そして彼らは、モータ制御を取り戻すまで、15分ごとに、麻酔後にそれらの回復を観察したラットを保ちます。また、回復期間中に角膜の乾燥を防ぐために、右目にテトラサイクリン軟膏を適用します。 </li></ol></li><li> 3日間連続して角膜cryoinjuryを繰り返します。 </li><li>前述のようにラットの眼の前房内にマリマスタットまたはbFGFのを提供するために、ラットを麻酔。一滴を植え付けます眼の痛みや瞬目反射を最小限に抑えるために、右眼に0.5％プロパラカイン塩酸塩。 </li><li>滅菌PBSで眼表面を灌漑。アイリスに上記と平行面でparalimbal透明な角膜で1mlシリンジに取り付けた30 G針を挿入することにより、手術用顕微鏡下で前房穿刺を行います。 </li><li>針のベベルアップを回し、少しいくつかの房水を排出し、眼圧を低下させるために角膜の傷を押し下げます。房内薬剤の0.02ミリリットルを注入します。静かに針引き抜きの間綿棒で針管を圧縮します。 </li><li>手術用顕微鏡下で指示された時点で、外部の目を撮影します。 </li></ol> 3.収穫ラット角膜ボタンと免疫染色<ol><li>ラットを安楽死させるために、安楽死室でそれらを配置し、毎分室容積の10から30パーセントの充填率で100％のCO 2を吹き込みます。以下のためのCO 2注入を維持します呼吸と色あせた目の色の欠如後の追加分。 </li><li>鋭利な刃で角膜輪部でのラットの目を貫通します。角膜輪部に沿って角膜ハサミで角膜をカットします。スライド上の角膜を平ら。角膜が丸まる場合には、追加の半径方向の切開を行います。 </li><li>室温で30分間、4％パラホルムアルデヒドを250μl、pH7.4で角膜を修正しました。 5分間、0.5％トリトンX-100250μlの透過性、及び30分間、10％のウシ血清アルブミンでブロックします。 </li><li> 1の希釈で4℃で一晩ABCに対する一次抗体を用いて角膜をインキュベート：抗体希釈液で200。 15分間、PBSで2回角膜を洗浄し、二次抗体でそれらをインキュベート：1時間室温で（1抗体希釈液中の100）。 4を2μg/ mlの細胞を覆うことによって細胞核を対比染色 &#39;、6-ジアミジノ-2-フェニルインドール、及び15分間、PBSで細胞を2回洗浄します。 </li><li>退色防止マウントソリューションで角膜をマウントします。オビ20X対物倍率でのレーザー走査型共焦点顕微鏡を用いて405と561 nmでの励起波長での免疫蛍光画像をテイン。 </li></ol>

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The authors have no competing financial interests to declare.

CECは、細胞増殖中EnMTを受けるそれらの傾向のために知られています。治療目的のためにEnMTプロセスを抑制するための戦略を開発するには、EnMTメカニズムの完全な理解が必要です。我々はEnMT、 インビトロ培養モデルおよびラット角膜内皮cryoinjuryモデルで 、すなわちウシCECを調査するために2モデルを説明しました。我々の結果は、両方のモデルでEnMTプロセスを実証しました。さらに、マリマスタットのEnMT抑制効果は、これらの2モデルが同じメカニズムを共有することを示唆し、両方のモデルで再現されました。 体外培養モデルにおけるウシCECは、操作のしやすさを提供しています。また、以前の研究17,18で採用インビトロ培養モデルにおけるヒトまたは霊長類CECと比較して、ウシの目が大きく、取得が容易であるため、より多くのCECを用意して。私たちの研究では、地元の食肉処理場から除核された牛の目を獲得しました。そのためFRESの<html>ウシ目の時間的性質は、収穫したウシのCECの正確な数は、眼あたり約1×10 5個の細胞で、変動の対象となります。これとは対照的に、正常な角膜における人間のCECの数は約3×10 5目あたりです。この数は、研究グレードの角膜に低く、移植後の残存角膜縁であっても低いです。採取手順の間の操作は、さらに得られた細胞の数を減少させます。また、ウシのCECは自発的にEnMTを増殖して受けることができます。したがって、 体外培養モデルにおけるウシCECはEnMTメカニズムを調査し、そのようなマリマスタットなどEnMT抑制化学物質を、スクリーニングに有用です。そのようなのCECの増殖能力などの種に関連した違い、懸念が残るものの、我々の結果は、ウシのCECのEnMTシグナル伝達経路がWnt /βカテニン18,19の活性化を含む人間のCEC、と同様であることを実証しました。 ウシのCECの単離の間にテント &quot;&gt;、デスメ膜を剥離することが重要である。いくつかのウシの目には、デスメ膜を剥離デスメ膜に過大な間質組織につながる、角膜実質との密着性を有することができる。さらに、トリプシン処理は、につながる可能性があり細胞の純度、したがって、実験結果に影響を与え、角膜線維芽細胞に変身間質角膜実質のリリース。私たちの研究では、顕微鏡下で慎重にデスメ膜を剥離した。さらに、細胞の純度を確保するために、我々は最初の6で回収された細胞を培養し細胞コンフルエンスまで10cmディッシュ。細胞の形状が六角形であっただけそれらがさらなる実験に使用しました。 さらに体外培養モデルにおけるウシCECの知見を実証するために、我々は以前の研究20,21で採用したラット角膜内皮cryoinjuryモデルを用いました。 cryoinjury後、ラットのCECはmanife、再生時のEnMTを受けましたWnt /βカテニンシグナル伝達の活性化はCECの再生中に種間で保存されていることを示す、アクティブβカテニンの核移行によってSTED。低CEC機能は明白な角膜浮腫につながるためまた、このモデルはのCECの機能を研究するのに有用です。このような超音波生体顕微鏡、前眼部光干渉断層撮影、および共焦点顕微鏡などの他の機器と組み合わせて、角膜の厚さを定量的に評価し、このようにCEC機能を反映することができます。 CECの再生を刺激し、創傷治癒後EnMTを抑制するために、我々は、マリマスタット注入しbFGFの房内注射を投与します。処置は、角膜浮腫の程度を減少させました。以前の研究では、このようなノッチ阻害剤21,22などのROCK阻害剤、およびEnMT抑制剤としてのCEC増殖刺激剤は、局所適用を使用していました。しかし、CECへの薬剤の浸透が横たわっていましたERは、治療効果23に影響を与える可能性角膜上皮および間質によって制限されます。局所適用とは対照的に、房内注入は、直接前房内に薬剤を導入することにより、角膜内皮への直接アクセスを可能にします。したがって、化学物質の増殖刺激とEnMT抑制効果がcryoinjury以下unbiasedly評価することができます。私たちの研究では、房内注入前に眼圧を下げるために前房穿刺を行いました。穿刺することなく、眼圧の急激な増加は、針管を通って角膜浮腫や虹彩のさえ脱出を引き起こします。静かに傷を密封し、眼内感染症を予防するには、針の離脱を助ける時に管を圧縮します。

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.
The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGF&#946;2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function. 
In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

<table><tbody><tr><td>trypsin</td><td>ThermoFisher Scientific</td><td>12604-013</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium and Ham&#039;s F12 medium</td><td>ThermoFisher Scientific</td><td>11330</td><td></td></tr><tr><td>fetal bovine serum</td><td>ThermoFisher Scientific</td><td>26140-079</td><td></td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>human epidermal growth factor</td><td>ThermoFisher Scientific</td><td>PHG0311</td><td></td></tr><tr><td>insulin, transferrin, selenium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>41400-045</td><td></td></tr><tr><td>cholera toxin</td><td>Sigma</td><td>C8052-1MG</td><td></td></tr><tr><td>gentamicin</td><td>ThermoFisher Scientific</td><td>15750-060</td><td></td></tr><tr><td>amphotericin B</td><td>ThermoFisher Scientific</td><td>15290-026</td><td></td></tr><tr><td>paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>111219</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787&amp;nbsp;</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>marimastat</td><td>Sigma</td><td>M2699-25MG</td><td></td></tr><tr><td>anti-active beta-catenin antibody</td><td>Millpore</td><td>05-665</td><td></td></tr><tr><td>anti-snail antibody</td><td>Santa cruz</td><td>sc28199</td><td></td></tr><tr><td>anti-slug antibody</td><td>Santa cruz</td><td>sc15391</td><td></td></tr><tr><td>goat anti-mouse IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11001</td><td>for staining of ABC of bovine CECs</td></tr><tr><td>goat anti-mouse IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11003</td><td>for staining of ABC of rat corneal endothelium</td></tr><tr><td>goat anti-rabbit IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11008</td><td>for staining of snail and slug of bovine CECs</td></tr><tr><td>antibody diluent</td><td>Genemed Biotechnologies</td><td>10-0001</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>laser scanning confocal microscope</td><td>ZEISS</td><td>LSM510</td><td></td></tr><tr><td>xylazine&amp;nbsp;</td><td>Bayer</td><td>N/A</td><td></td></tr><tr><td>tiletamine plus zolazepam</td><td>Virbac</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>proparacaine hydrochloride ophthalmic solution</td><td>Alcon</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>0.1% atropine</td><td>Wu-Fu Laboratories Co., Ltd</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>0.3% gentamicin sulfate</td><td>Sinphar Group</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>basic fibroblast growth factor</td><td>ThermoFisher Scientific</td><td>PHG0024</td><td>clinical drug&amp;nbsp;</td></tr></tbody></table>

in vitro and in vivo models to study corneal endothelial-mesenchymal transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

ウシのCECの単離後、細胞をインビトロで培養した。 図1は、ウシのCECの位相コントラスト画像を提示します。コンフルエンスの細胞の六角形は、細胞が細胞単離の間、角膜間質線維芽細胞によって汚染されていないことを示した。 図2に示された時点でABC、カタツムリ及びナメクジに対する抗体を用いて行った免疫染色を示します。 in vitro培養における表現型の変化とは別に、ABCとEMTレギュレータの対応する核内移行を観察した。 図3は、in vitroで培養されたウシのCECのEnMTプロセスに、マリマスタット、広域スペクトルのMMP阻害剤の効果を示す。 図図4は、房内注入しcryoinjury後のラットの外部目の写真を含んでいる。 図5は、Rの免疫染色を示しています<html>房内注射、続いcryoinjury後ABCに対する抗体を用いて行った角膜ボタンで。 ABCの核移行は、PBS群で観察された有意なWnt /βカテニンシグナルとEnMTプロセスの活性化を示し、bFGFの群において増加しました。マリマスタット注射したbFGFの房内注入した後、ABCの核染色は、マリマスタットのEnMT抑制効果を示唆し、減少しました。 <img alt="図1" src="/files/ftp_upload/54329/54329fig1.jpg" /> 図1：in vitroで培養されたウシのCECは、培養プレート上に播種された後、ウシのCECが最初に現れたの位相コントラスト画像は、線維芽細胞様の日3と6には、彼らは= 9日スケールバー上の完全なコンフルエンスに達した時に、より六角形になりました50μmです。 3反復の代表的な画像。4329fig1large.jpg &quot;ターゲット=&quot; _空白 &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図2" src="/files/ftp_upload/54329/54329fig2.jpg" /> 図2：アクティブβカテニン：in vitroで培養されたウシのCECウシのCEC、ABC、カタツムリ、およびスラグの核移行のin vitro培養中に14日ABCを通じて検出されたの免疫染色 。スケールバー=100μmです。 3反復の代表的な画像。 <a href="https://www.jove.com/files/ftp_upload/54329/54329fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54329/54329fig3.jpg" /> 図3：付きまたは異なる細胞合流レベルでマリマスタットなしのin vitroで培養ウシのCECの免疫染色免疫染色悪魔ABCは、カタツムリ、およびスラグを持つか、しかし3日目にマリマスタットの10μMのない牛のCECの核内で明らかになったことをtrated、マリマスタットは大幅に9日目ウシのCEC上の核ABC、カタツムリの染色、およびスラグを縮小しました完全にコンフルエントになりました。スケールバー=100μmです。 3反復の代表的な画像。 <a href="https://www.jove.com/files/ftp_upload/54329/54329fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54329/54329fig4.jpg" /> 図4：示された時点でのラットの外部目の写真cryoinjury後 3日間連続cryoinjury後、ラットは、6日目、3日目にPBSまたは50 ngの/ mlのbFGFの0.02ミリリットルの房内注入に供した、0.02ミリリットルの。 PBSは、他の2 grouに注入したのに対し、10μMマリマスタットは、bFGFを/マリ・グループに房内に注入しましたPS（各群n = 9）。外部の目の写真は、bFGF単独と比較してマリマスタットさらに還元角膜浮腫のに対し、bFGFを注射した後に減少した角膜浮腫を明らかにした。各群n = 9。スケールバー= 1mmである。 <a href="https://www.jove.com/files/ftp_upload/54329/54329fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図5" src="/files/ftp_upload/54329/54329fig5.jpg" /> 図5：9日目に収穫したラット角膜ボタンのラット角膜ボタンの免疫染色免疫染色は、PBS群でABCの小さな核染色を明らかにしました。 bFGFのグループでは、かなりのbFGF /マリ群において減少したABCの大規模な核染色は、ありました。スケールバー=100μmである。 <a href="https://www.jove.com/files/ftp_upload/54329/54329fig5large.jpg" target="_blank">THIの拡大版をご覧になるにはこちらをクリックしてください。</a>sのフィギュア。

Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

ウシ角膜内皮細胞の初代培養は、角膜内皮間葉移行のメカニズムを調べるために使用しました。さらに、ラット角膜内皮cryoinjuryモデルは、in vivoで角膜内皮間葉移行を示すために使用されました。

in vitroおよびin vivoモデルで角膜内皮間葉転換を研究します

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

in-vitro-vivo-models-to-study-corneal-endothelial-mesenchymal

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

7.9K ビュー.  Far Eastern Memorial Hospital. ウシ角膜内皮細胞の初代培養は、角膜内皮間葉移行のメカニズムを調べるために使用しました。さらに、ラット角膜内皮cryoinjuryモデルは、in vivoで角膜内皮間葉移行を示すために使用されました。 

ビデオ: In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

角膜組織工学: In Vitroのモデル人間の角膜の神経間質相互作用

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

発生生物学

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

ネズミ角膜移植：固体臓器移植の最も一般的な形態を研究するためのモデル

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

医学

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜マイクロアッセイ：マウス眼における血管新生のモデル

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

神経科学

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

生体物質膜上におけるヒトおよび羊角膜内皮細胞層の増殖

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

生物工学

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

分割角膜ボタンを用いてのブタ角膜内皮器培養モデル

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

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 ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

角膜上皮の機械的および化学的損傷のためのEx Vivoおよびin vivo動物モデル

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

成長因子 β シグナルを変換することによって誘導される血管内皮間葉移行の分子解析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

生物学

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 ...