この研究は網膜変性疾患基礎戦いの盲目の失明を防ぐため、ニューヨーク、www.rpbusa.org、およびグレーター ニューヨーク センター研究によってサポートされて www.blindness.org。著者は、この原稿の彼女の批評の Luanna バーソロミュー、博士を感謝したいです。

次のプロトコルは、ヘルシンキ宣言とイェール大学人間研究倫理委員会ガイドラインの原則への付着で実行されます。
1. 目人間のドナーを調達
<ol><li>国立病研究インターチェンジから (NDRI, フィラデルフィア, ペンシルバニア) 人間のドナーの地球儀を取得します。プロジェクトによって、ドナーの目の 3 つ以上のペアを各実験群に備えます。</li>
	<li>10 h 以内ドナー目と 48 h事後内船を摘出します。その後、滅菌ダルベッコ改変イーグル培地 (DMEM) 文化培地氷の上で研究室に目を転送します。以前報告された研究は、ドナーの目が収穫され、生物学的特徴とブルッフ膜25,26,29の活動、このファッションの保持で輸送を実証しています。</li>
</ol>2. 収穫人間ブルッフ膜の外植体します。
<ol><li>場所は 1.5 × 1.5 インチ ガーゼにそれぞれの目に浸した 100 mm ディッシュ用と 3 mm 強膜切開を角膜輪部の後方に使用高級刃はさみで炭酸ガスに依存しない DMEM メディアと円周方向のどちらの方向に拡張します。</li>
	<li>作る鋸状縁に大形環状切開 (硝子体に貫通) 1 mm 前後。削除し、前眼部 (角膜、レンズ、およびアイリス)、硝子体と網膜を破棄します。</li>
	<li>微細手術用ハサミを使用して強膜と脈絡膜の間に 4 つの放射状切開と複雑な脈絡膜/ブルッフ膜を引き裂くことを避けるために世話をしながら、視神経に周囲から強膜をはがします。</li>
	<li>次に、鋸状縁に沿って脈絡空間を介して環状切開をして後方の強膜から網膜色素上皮-脈絡膜脈絡膜複合体の皮をむきます。</li>
	<li>ダルベッコ リン酸緩衝生理食塩水 (DPBS) 60 × 15 mm ポリスチレン シャーレ DPBS (図 1) で 3 回洗い、その後、室温で 20 分間で 0.02 M アンモニア水を外植体をお風呂でネイティブの網膜色素上皮細胞を削除します。</li>
	<li>二酸化炭素自由な媒体の膜を直面している各植の基底層での 0.2 μ m 孔 unlaminated、疎水性の 65 μ m 厚ポリテトラフルオロ エチレン膜上、ブルッフ膜複雑な植を浮かんでいます。</li>
	<li>ガラスフリット ガラス サポートにガラス微量真空フィルター ホルダー システムでブルッフ膜植ポリテトラフルオロ エチレン膜を配置します。</li>
	<li>微細鉗子、ブルッフ膜基底ラミニン側に触れないように世話テフロン加工で脈絡膜側からカールされた端を平らにします。</li>
	<li>アガロース (DPBS 4%) の 15 mL を加熱し、37 ° C に冷却してからその後外植体がフラットに残ることを保証する穏やかな吸引を適用中に脈絡膜側から、ブルッフ膜脈絡膜複合体液状化・ ゲルの 15 mL を注ぐ。Agarose を固めるし、ポリテトラフルオロ エチレン膜を離れて皮をむくに 2-3 分間、4 ° C で組織を維持します。</li>
	<li>ブルッフの膜を固定する皿の底に温かい液体 agarose が付いて並んでされている (上向きブルッフ膜) と 60 × 15 mm ポリスチレン細胞培養皿の中、ブルッフ膜植 (凝固 agarose のゲル内で、直径 1.5 インチ) を配置します。上向き植基肥ラミニン層で井戸の底の外植体します。</li>
	<li>Agarose を培養皿で、ブルッフ膜外植体を安定させるために室温で 2-3 分以内で固めます。文化の外植片、ブルッフ膜カバー DPBS 皿し、将来の使用のための 4 ° C で料理を保存します。図 2 aは、60 × 15 mm 培養皿の外植体分離ブルッフ膜を示しています。</li>
	<li>目的のカルチャ システムが、96 ウェル プレート、ラミニン側で平らなテフロン シートでのブルッフ膜植 (凝固 agarose のゲルで、直径 1.5 インチ) 上に置きます。</li>
	<li>5 か 6 をカットのトレフィンを使用、ブルッフ膜複合体から 6 ミリメートル円形ボタン外植体し、96 ウェル プレートの無処理のポリスチレンの 37 ° C で 4% の agarose に一箇所します。Agarose を固める各井戸の底で、ブルッフ膜植 (ボタン) を修正するために室温で 2-3 分以内。 注:図 2 bは、96 ウェル プレートで trephined ブルッフ膜のボタンを示しています。通常、9-11 のボタンまたは植がそれぞれの眼から収穫できます。</li>
</ol>3. 外植体人間ブルッフ膜の網膜色素上皮 (RPE) 細胞の培養
<ol><li>ドナー目の受領、DPBS と眼組織をきれいに。タッチ ポイント (pars プラナ) 下腔へ環状切開アイリス強膜の下の 5 mm を作る。前眼部、硝子体液、網膜を破棄します。</li>
	<li>ブルッフの膜と網膜色素上皮シート、冷たい DPBS 含むアイカップを洗います。もはや目の各サンプルに対して 10 分以上のための 0.25% トリプシンの 5 mL と網膜色素上皮細胞を分離します。DMEM 15% ウシ胎児血清 (FBS) 50 mL のチューブで 37 ° C で暖められた完全な媒体の 25 mL とトリプシンの反応を消します。</li>
	<li>24 の ° C で 10 分間 200 × g で細胞を遠心し、DMEM 完全培地 5 mL にペレットを再懸濁します (15 %fbs)。</li>
	<li>セルをカウントし、それぞれの様々 な高齢者ブルッフ膜外植体 (ボタン) 無血清最低限不可欠なメディア (MEM) 200 μ L 中の 96 ウェル プレートに抗生物質を含む基底膜に 15,000 の実行可能な網膜色素上皮細胞を播く (100 IU/mL ペニシリン G、100 mg/mL ストレプトマイシン、ゲンタマイシン、5 mg/mL、2.5 mg/mL アムホテリシン B)。この密度でめっき 20 μ m の細胞径を仮定してメッキ面積の約 15% をカバーする網膜色素上皮細胞になります。</li>
	<li>いう細胞密度、段階的に増加細胞挙動に及ぼす各パラメーターによって 100% 合流ボタンの上に細胞をプレートします。</li>
	<li>細胞は、加湿雰囲気 95% air/5% CO2 37 ° C での 24 h の外植体に付ける優しく温かみのある完全な DMEM に媒体を変更します。</li>
</ol>

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作者が宣言する商業開示をあります。

高齢者人間ブルッフ膜 (細胞外マトリックス) は、AMD の病気の進行に貢献し、従ってこの変更された行列が網膜色素上皮細胞機能障害に貢献する方法を理解する必要があります。AMD の加齢変化を検討するための十分な動物モデルの欠乏を与えられて、病気の効果を模倣のモデル システム前のヴィヴォは病態生理を理解するための貴重なツールとして使用できます。本稿で説明している手法を使用して、一貫してブルッフ膜外植片や培養網膜色素上皮細胞、主、不死化、または幹細胞由来網膜色素上皮細胞を含む人間を分離できます。
前述のように、人間のブルッフ膜外植片は、NDRI から供給されます。NDRI ドナー目が許容されるための基準セットを指定すると、プロトコルを設立します。たとえば、ドナーは知られている網膜目の病気をしてはならない、死後後 10 時間以内取得目/地球儀、地球儀が (氷の上の夜通しのパッケージ配達) を経由して死後後 48 時間以内研究室に到着します。適切な年齢範囲と、例えば研究の統計解析に必要な目数を指定します。、20-49 歳の年齢層と 50-89 歳の年齢の間のドナーからの 5 つの世界ペア間ドナーからグローブの 5 つのペア。
通常の平均使用可能な地球儀の 10 ペア毎月目地球儀の可用性が異なります。目パッケージは、基本的な匿名化したドナー情報と共に到着: 年齢、競争、死、死の原因の時、既往歴 (病気一覧または併存疾患) 簡単な。
最も重要なは、目の硝子体、網膜色素上皮-脈絡膜脈絡膜複合体の分離を可能にする眼の除去が必要です人間ブルッフ膜外植体を収穫します。一度、ブルッフ膜が分離されている網膜色素上皮細胞をさまざまな濃度で無細胞植にシードし、下流の実験のために培養できます。記載としては重要な亂の表面が機械的に損傷を防ぐため表面を触れていないが直面して-を確認する分離ブルッフ膜の向きに注意を払って準備と手続き型の処理、細胞外マトリックスの表面構造。
ブルッフの膜植システムを使用している場合、個々 の供給ゲノム背景など下流の実験の結果、ブルッフの膜の年齢や、ブルッフの死後の時間に影響を与える可能性があります。 いくつかの変数があることを指摘する必要があります。膜とブルッフの膜、すなわち場所。 中枢または末梢、ブルッフ膜外植体の一部。他のヒト組織研究と統計力を持つし、一致ドナー眼年齢に応じて適切なドナー目サンプル数を募集する必要があります。アイバンクから目を受け入れるための厳格な基準を確立することは重要なパラメーターです。10 h 未満死後を除を死の後 24-48 時間以内、ブルッフ膜作製を収穫することができます目を受け付けます。視神経サイト向きのマーカーとして使用されるかもしれないし、の同じ場所を使用実験および研究グループを制御します。これらの措置を講ずること、実験的変動を最小限に抑えることができます 1 つ。
要約すると、網膜色素上皮-脈絡膜脈絡膜複合体および年齢や病気によって影響の理解は AMD の病態への貢献を理解する重要です。前のヴィヴォ技術採用モデル システムは、人間のティッシュを使用してこれらの効果を調査する貴重なツールです。ここで、高齢者ブルッフ膜の関連性の高いex vivoモデルとして人間のドナー植を採用する方法論について述べる。この手法マトリックス内の構造の変化を調査する研究ツールは RPE を重ねることを影響することが、高齢者や病気にかかった人間ブルッフ膜を使用することができます添付ファイル、増殖、および遺伝子発現25を含むセル動作,27. 同じような前のヴィヴォモデル システムの開発に成功、AMD の理解を深めるし、新規治療方法の開発を促進します。

Nitrosative 酸化ストレスなど、多くの要因によって引き起こされる人間ブルッフ膜の構造の変更を加齢に伴う網膜色素上皮 (RPE) 細胞し、に貢献の機能複数の有害な効果を発揮する、加齢に伴う黄斑変性症 (AMD)1,2,3,4,5,6,7,8,9の病理.高度な萎縮性 AMD または地理的な萎縮細胞補充療法を検討するとき、治療は網膜色素上皮細胞の萎縮のベッドの上に細胞移植を必要可能性が高い。人間ブルッフ膜内の年齢関連の変更することができます悪影響を細胞外マトリックス6,9,10,11に被害を与えられた移植の網膜色素上皮細胞移植の成功,12,13,14,15,16,17,18,19,20,21,22. ブルッフ膜生物学およびマトリックス内の構造の変化は AMD への進行に貢献調査人間が病の病理を理解することが重要です。従って、高齢者や病気にかかった人間ブルッフ膜のex vivo収穫を記述するプロトコルを開発する加齢に伴う目のフィールドの調査官の重要な必要性があります。
歴史的、地理的な萎縮など動物23歳人間ブルッフの膜モデル加齢に伴う疾患に、難しかったです。この難しさは、齧歯動物およびモデリング、病だけでなく、ほとんどの脊椎動物24黄斑の欠如のために頻繁に使用される他の種と比較して人間の寿命を含む複数の要因から発生します。記載方法の利点は、高齢者や病気の個人のポスト mortem 目から抽出されたブルッフ膜の上に直接セルをテストできることです。この記事の全体的な目的は、高齢者や病気にかかった人間ブルッフ膜のex vivoモデルの詳細な方法を提供する、人間ブルッフの分離人間のドナーから外植体膜などの細胞網膜色素上皮の播種下流の実験。このモデルは、RPE 細胞機能と病理20,25,26,27,28損傷細胞外マトリックスの影響を検討する関連するモデルとして使用できます。

<table><tbody><tr><td>Human donor globes</td><td>National Disease Research Interchange, NDRI</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Thermo Fisher Scientific</td><td>11995-065</td><td></td></tr><tr><td>Carbon-dioxide independent media</td><td>Thermo Fisher Scientific</td><td>18045-088</td><td></td></tr><tr><td>60-mm polystyrene petri dish</td><td>Corning Inc.</td><td>351007</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline, PBS</td><td>Thermo Fisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Hydrophobic 65 &amp;micro;m-thick polytetrafluoroethylene membrane with 0.2 &amp;micro;m pores</td><td>Merck Millipore</td><td>JGWP04700</td><td></td></tr><tr><td>Fisherbrand glass microanalysis vacuum filter holder system&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>09-753-102</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A2576-5G</td><td></td></tr><tr><td>35 &amp;times; 10mm culture dish</td><td>VWR International</td><td>25373-041</td><td></td></tr><tr><td>Trephine</td><td>Accutome</td><td>AM0570 60</td><td></td></tr><tr><td>96 well plate</td><td>Corning Inc.</td><td>3595</td><td></td></tr><tr><td>Minimum essential media (MEM)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11095-080&amp;nbsp;</td><td></td></tr><tr><td>Penicillin G</td><td>Sigma-Aldrich</td><td>P3032-1MU</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich</td><td>S6501</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1914</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>1397-89-3</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma-Aldrich</td><td>1336-21-6&lt;br/&gt; &amp;nbsp;</td><td></td></tr></tbody></table>

culturing of retinal pigment epithelial cells on an ex vivo model of aged human bruch's membrane

別にビタミンや抗酸化物質加目病論文での推奨の「乾燥」や萎縮性加齢による黄斑変性症 (AMD) の場合の 90% を表すため有効な治療法はありません。療法が低速または地理的な萎縮 (GA) の発達を遅らせる必要し、ブルッフ膜の病理を理解することは、このプロセスの一部です。人間ブルッフ膜の変化は、網膜色素上皮 (RPE) の損傷に貢献する細胞 AMD の進行を前します。AMD を勉強するための十分な動物モデルの欠如を考えると、高齢者の人間ブルッフ膜のex vivoモデル提供から網膜色素上皮細胞の挙動を調べるための便利なツールが不死化し、RPE 線と同様、主細胞由来の多能性幹細胞 (Ips)。ここでは、添付ファイル、アポトーシスと増殖、感光体の外側を phagocytize する能力を含む人間のドナーから収穫された人間のブルッフ膜植でシード RPE 細胞の挙動の影響を確認することができます詳細な方法を提案します。セグメント、極性、および遺伝子発現の確立。この試金は、網膜色素上皮細胞と細胞外マトリックスの高齢者/侵害でシードの機能特性を評価するために高齢者ブルッフ膜のex vivoモデルを提供します。

このプロトコルは正しく実行されると、極性と外血液網膜関門の確立、成長因子とサイトカインの偏光の分泌によって高齢者や病気にかかった人間ブルッフ膜の網膜色素上皮細胞の機能に及ぼす影響を決定できます。視細胞視細胞外節の貪食。
私たちは、高齢者人間ブルッフ膜の病気の表現型を示すデータを生成しています。たとえば、網膜色素上皮細胞の養殖人間ブルッフ膜外植体減少 RPE 細胞付着 (図 3) を高齢者。高齢者人間ブルッフ膜上で培養した網膜色素上皮細胞する能力を減らすこれらの細胞の視細胞外節 (ROS) を phagocytize 重要な RPE 関数 (図 4)。さらに、アポトーシス細胞を識別および前仕事26に記載されているニック dUTP エンドラベル TUNEL 染色トランスフェラーゼ遊離を使用してこれらのボタン植に比較が可能します。マイクロ アレイ技術を用いた、高齢者や病気にかかった人間ブルッフ膜の網膜色素上皮細胞の遺伝子発現プロファイルに及ぼす影響を判断できます。若い個人 (図 5)27から植と比較して高齢者から人間ブルッフ膜上で培養したときに、網膜色素上皮細胞の遺伝子発現を変更することを確認しました。統計分析、グループごと、少なくとも 3-5 人間のドナーから植が使用されます。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57084/57084fig1v2.jpg"> 図 1。ドナーの目から人間ブルッフ膜の分離の略図。人間ブルッフ膜、強膜、眼、網膜、硝子体を削除することによって収穫され、ネイティブ網膜色素上皮 (RPE)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57084/57084fig1v2large.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/57084/57084fig2.jpg"> 図 2。分離培養皿で人間ブルッフ膜 (BM).(A)人間ブルッフ膜植 (凝固 agarose のゲルで、直径は 1.5 インチ各) の模式図に 60 × 15 mm ポリスチレン細胞培養ディッシュ (BM 顔アップ) を皿の底に温かい液体アガロースでいっぱい。いくつかの 6 mm 円形ボタン (植) に trephined されています(B)人間のブルッフ膜植わーを置かれた 96 ウェル プレートの無処理のポリスチレンの 37 ° C で 4% アガロースゲルの各。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57084/57084fig2large.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/57084/57084fig3.jpg"> 図 3。網膜色素上皮 (RPE) 細胞若くて古い人間ブルッフ膜 (BM) の付着率。脈絡膜は、若いから外植体人間にプライマリの網膜色素上皮細胞を播種 (< 50 歳、n = 3) 以上 (> 70 歳、n = 5) ドナー 3 週間目。MTT セル実行可能性の試金によって添付ファイルのセル速度を測定しました。古い人間ブルッフ膜は網膜色素上皮細胞の添付率を 24% 減 (8.54 +/-若い BM 100 ± 1.44 古い BM 76.65 対東南東南)。p < 0.05、東南標準エラーを =。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57084/57084fig4.jpg"> 図 4。網膜色素上皮 (RPE) 細胞の貪食能は人間ブルッフ膜 (BM) の年齢を受けます。古い人間がブルッフ膜外植体上で培養したプライマリの網膜色素上皮細胞は視細胞外節 (ROS) を phagocytize する能力が低下 (9.3 ± 若い BM 873 東南、n = 25.4 ± 古い BM 608 対 5 東南、n = 5)。p < 0.01、東南標準エラーを =。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57084/57084fig5.jpg"> 図 5。網膜の顔料の若い上に培養した各サンプルの発現上皮 (RPE) 細胞遺伝子または古い人間ブルッフ膜外植体します。主な網膜色素上皮細胞上に発現する遺伝子の数のほか、散布があった若い人間ブルッフ膜と古い (6201 ± 388 6439 ± 80、対それぞれ);5 植は、年齢グループごとのテストされました。Cai、すべての作家の完全な許可は、Hら。27.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57084/57084fig5large.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 Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane at JoVE.com

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

このプロトコルの目的は、高齢者や病気にかかった人間ブルッフ膜の網膜色素上皮 (RPE) 細胞の培養を示すことです。このメソッドは、侵害された細胞外のマトリックスの RPE 細胞の挙動を研究に適しています。

高齢者人間ブルッフ膜のEx Vivoモデルの網膜色素上皮細胞の培養

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

culturing-retinal-pigment-epithelial-cells-on-an-ex-vivo-model-aged

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

10.1K ビュー.  Yale School of Medicine. このプロトコルの目的は、高齢者や病気にかかった人間ブルッフ膜の網膜色素上皮 (RPE) 細胞の培養を示すことです。このメソッドは、侵害された細胞外のマトリックスの RPE 細胞の挙動を研究に適しています。

ビデオ: Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

網膜色素上皮生理学と病態の研究のための実験的モデル

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

神経科学

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center&#160;of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch&#8217;s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch&#8217;s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch&#8217;s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch&#8217;s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch&#8217;s membrane biochemistry.

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

ヒトドナー目からブルッフ膜の濃縮

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

無条件網膜色素上皮を用いた、オルガマウス網膜の長期無血清栽培

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新しいヒトの言語性の高いレティナル文化技術の特徴付け

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

医学

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

ヒト以外の霊長類モデルにおける変性網膜疾患の網膜色素上皮移植

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

培養初代マウスRPEの特性評価と形態学的ダイナミクス

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

著者スポットライト:網膜症研究のための成体マウスからの一次網膜色素上皮細胞の効率的な単離

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

マウス網膜色素上皮からの一次電池文化

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

生物学

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

非ウイルス遺伝子治療のための哺乳動物一次色素上皮細胞の単離、培養、遺伝子工学

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

高齢者人間ブルッフ膜のEx Vivoモデルの網膜色素上皮細胞の培養

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網膜色素上皮生理学と病態の研究のための実験的モデル

ヒトドナー目からブルッフ膜の濃縮

無条件網膜色素上皮を用いた、オルガマウス網膜の長期無血清栽培

新しいヒトの言語性の高いレティナル文化技術の特徴付け

ヒト以外の霊長類モデルにおける変性網膜疾患の網膜色素上皮移植

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

マウス網膜色素上皮からの一次電池文化

非ウイルス遺伝子治療のための哺乳動物一次色素上皮細胞の単離、培養、遺伝子工学