Name: development of a refined protocol for trans-scleral subretinal transplantation of human retinal pigment epithelial cells into rat eyes
Uploaded: 2017-08-12T13:00:00.000+00:00
Duration: 12 min 45 s
Description: Watch this Scientific Journal Video about Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes at JoVE.com

我々 は RPE 細胞の準備のため手術とスーザン ボーデンに彼女の援助のためのパテ レダーマンを感謝したいです。我々 はまた、このプロジェクトのための資金の NYSTEM C028504 を認めます。ジャスティン D. ミラーは NIH によってサポートされて F32EY025931 を付与します。

動物を含むすべてのプロシージャは、機関動物ケアおよび使用委員会 (IACUC)、アルバニーのニューヨークの州立大学、によって承認されています。
1. 事前注入準備
<ol><li>
HRPESC RPE の細胞懸濁液の調製 注: 滅菌のティッシュ文化フードのすべての次の手順を実行し、基本的な生殖不能の技術と知識が必要です。<ol>
<li>主な hRPE 細胞を分離および 24 ウェル プレート12に培養細胞を 50 から 90 年間人間のドナーから目が高齢者します。通路 1 細胞を凍結、融解必要に応じて文化通路 2 (P2) 細胞の注入の 4-5 週間 (図 1 a) の。</li>
		<li>培地12を外し、軽く 500 μ L で 2 回リンス井戸で加温ダルベッコ リン酸緩衝生理食塩水カルシウム & マグネシウムなし X 1 (1 DPBS CMF x) 1 を追加することによって X DPBS-CMF 井戸 1,000 μ L ピペットを使用し、真空を使用してそれを削除することに。</li>
		<li>各ウェルに 300 μ L トリプシン/DNAse を追加します。トリプシン/dn (0.25% トリプシン-EDTA の 1 mL あたり 4 区 DNase) セルを分離する 37 ° C で 4 分で hRPESC 網膜色素上皮細胞を孵化させなさい。</li>
		<li>彼らを切り上げているかどうかを顕微鏡下で細胞を確認します。まだ切り上げ彼らない場合さらに 2 分間トリプシン/DNase のセルをインキュベートし続けます。</li>
		<li>セルが切り上げられます、一度カップ刻んだ井戸から剥離細胞とトリプシン/DNase を不活化する、予め温めておいた培の等量と 15 mL の円錐管に剥離細胞を含むトリプシン/DNase を転送する 1,000 μ L ピペットを使用します。</li>
		<li>予め温めておいた 1 と井戸をリンス優しく特に; 井戸の縁の周りを上下、ピペッティングにより X DPBS CMF以前の円錐管にこれらの細胞を追加します。</li>
		<li>細胞のペレットへの 4 ° C で 5 分間 286 x g で円錐形の管を遠心します。</li>
		<li>上澄みを除去し、培地 1 mL の細胞を再懸濁します。</li>
		<li>診断を使用してセルをカウントします。</li>
		<li>細胞のペレットへの 4 ° C で 5 分間 286 × g で遠心分離機します。</li>
		<li>上澄みを除去し、細胞に滅菌バランスの取れた塩ソリューション (BSS) 50,000 細胞/μ L で (網膜注入中に 1 μ L のボリュームの 50,000 セルを配信) に再懸濁します。</li>
		<li>1.7 mL 遠心チューブに氷-水混合物の注入に使用されるまで最終的な細胞懸濁液 (CS) を転送します。</li>
	</ol>
</li>
	<li>
セル インジェクターの準備
	<ol>
<li>注射器とインジェクターを組み立てるにはしっかりとネジに滅菌 33 ゲージ ベベル針を挿入します。</li>
		<li>5-6 回 100% エタノールでインジェクターをフラッシュします。</li>
		<li>5-6 回の 70% エタノール噴射装置をフラッシュします。</li>
		<li>5-6 回 BSS とインジェクターをフラッシュします。</li>
		<li>(図 1 b) 解剖顕微鏡下で針の先端から 600 μ m の位置にある黒のマーカーペン滅菌とインジェクター針をマークします。</li>
		<li>注射用マイクロマニピュレーターにインジェクターを配置します。</li>
	</ol>
</li>
</ol>2. 網膜注入
<ol><li>
外科領域と動物の準備
	<ol>
<li>4-5 週齢 RCS ラット (60-100 g) の重量を量るし、イソフルラン蒸気の配信システムを使用してそれを麻酔します。 注:誘発するには、麻酔は 5% にイソフルランの流量を維持します。足を押すことによって麻酔の深さを確認し、手術中に麻酔維持のための 2-3%、流量を減らします。</li>
		<li>滅菌手術ドレープ、滅菌手術領域を設定する解剖顕微鏡のステージの下に、加熱パッドを配置します。</li>
		<li>外科領域にラットを転送し、ラットを麻酔を維持するためにイソフルラン システムに接続されている鼻の円錐形に配置します。</li>
		<li>ガーゼでラットの体をカバーしてください。完全麻酔を確認するラットのつま先をピンチします。</li>
	</ol>
</li>
	<li>
顕微鏡下強膜網膜注入
	<ol>
<li>ラットの unoperated 目に目の潤滑剤の滴を適用します。</li>
		<li>注射、外科医の右手に向かって頭とその外科医の方のための天井に直面して左眼の右側にラットを配置します。</li>
		<li>小さなハサミで目をカバーするひげをトリムします。</li>
		<li>左眼の時間的側面から目の洗浄量を点滴し、目を洗うに綿棒で鼻側に過剰を収集します。</li>
		<li>それぞれの滴を適用することによって投与後 10 月受験 (たて 10% フェニレフリンから手術の日にそれ 0.9% 生理食塩水で希釈することによって作られる) 1% 2.5% とトロピカミド フェニレフリンで瞳孔を散大させます。</li>
		<li>4-6 回目の周囲の皮膚を軽く引いてするまぶたを開き、目は若干れ角膜輪部の後方の領域にアクセスするため、突出しています。</li>
		<li>目の洗浄の適用ドロップ、目の潤いを保ちます。</li>
		<li>優しく (1.1. 12 のステップで準備) CS をカップ刻んだし、1.2 μ L CS とインジェクターをロードします。余分な 0.2 μ L が注入逆流を補うために使用されます。 注:について私たちの測定に基づく 5,000-8,000 セルはセル損失の約 10-16% に相当する 50,000 の細胞/μ の注射で逆流で失われ、0.2 μ L CS の余分をこのセル損失を補償するために注入されました。</li>
		<li>満ちている CS マイクロマニピュレーターにインジェクターを配置 (またはそれを保持するアシスタント) 垂直として RPE 細胞傾向にある懸濁液で簡単に沈みます。</li>
		<li>目にプロパラカイン 0.5% (ローカル局所麻酔薬) のドロップを適用し、綿アプリケーター付き過剰を削除します。 注:この手順は、角膜反射を抑制するし、目が後続の手順の実行中に点滅するを防ぐ必要があります。</li>
		<li>結膜、角膜の後方をグリップし、鼻、目を回転させる「テント」結膜を持ち上げて鉗子を使用します。</li>
		<li>結膜に小さな開口部を確認し、基になる強膜を公開「テント」上部をカットするはさみを使用します。</li>
		<li>鉗子を使用して、残り結膜角膜マージンの端をつかんで、瞳孔軸テーブル トップ (図 1) を基準にして約 30 度の角度で、鼻、目を回転します。針挿入時反力を提供するために、最適な角度で目を維持するために、結膜縁の継続的な把握が必要です。</li>
		<li>細胞の注入のための下穴をするためには、滅菌ベベル 31 ゲージ インスリンの針の先端位置は 1,200-1,500 μ m 直面している先端の開口縁の後方で。</li>
		<li>10-15 度 (目的の注射部位の虚数平面に接線) 強膜上にあるインスリンの針の角度を調整します。約 500 μ m の針の深さに強膜・脈絡膜複合体にゆっくりと浸透します。色素性のラットで針の端が「消滅」色素性の脈絡膜の下にあります。ここで使用されるインスリンの針のブランド、針の先端から面取りまでの距離は 500 μ m です。</li>
		<li>慎重に撤回するインスリンの針 (血の非常に小さい胸水が見られるかもしれない)。</li>
		<li>過度の出血が認められる場合、目槍は、必要に応じて、穴をオフに適用できます。継続的な出血後槍アプリケーション容器が破損しています。</li>
		<li>上下強膜の局所的な面を基準にして約 10-15 度の角度に直面して開口をパイロット穴に挿入、RPE 細胞ロード インジェクター針をガイドします。</li>
		<li>インジェクター針を網膜の領域にアクセスする約 500 μ m の深さにパイロット穴にそっと挿入します。黒のペンのマークの端と針が (図 1と1 D) 色素性の脈絡膜で覆われているポイントの間 100 μ m のマージンについて必要があります。</li>
		<li>優しく細胞 (約 1.2 μ L) の適切な量を注入する注入器注射器のプランジャーを押し下げるにアシスタントを頼みます。プランジャーを押すとアシスタントとしていくつかの反力を提供する準備ができてください。 注:アシスタントの前に模擬練習は外科医とこのステップに必要な経験を持つアシスタントの両方を提供できます。</li>
		<li>ペンで視覚的に焦点を当てながらエッジをマーク、25-30 秒の場所にインジェクターを保持、インジェクターをゆっくりと上げます。少量の逆流がよく観察されます。 注:逆流を認められなかった場合で、ケナコルト硝子体内注入をされてありますいる可能性があります。逆流シールや、強膜の下に充填セルを介して場合は、注入が浅すぎていた。</li>
		<li>3 回滅菌目洗うと注射部位から細胞排出を洗うし、綿アプリケーター付き過剰を収集します。</li>
		<li>運営の目に目の潤滑剤の滴を適用し、ラットを移植細胞の場所と網膜の胞のサイズを検討する 10 月駅に転送します。</li>
	</ol>
</li>
</ol>3. 後注入治療
<ol><li>
抗炎症、痛みの緩和治療
	<ol>
<li>痛みを軽減する皮下生理食塩水で 0.1 mg/kg 体重でブプレノルフィンを注入します。</li>
		<li>腹腔内注入 (i. p.) 炎症制御による生理食塩水で 1.6 mg/kg 体重でデキサメタゾンを注入します。</li>
	</ol>
</li>
	<li>
動物の回復
	<ol>
<li>ラットを体温を維持する熱ランプの下で回復ケージに戻ります。</li>
		<li>運営の目には出血の兆候を確認します。</li>
		<li>麻酔が出てくるようにラットを観察します。</li>
		<li>回復した動物を新鮮なケージに戻る手術カードでケージをフラグし、毎日の苦痛、眼出血、角膜混濁の兆候を監視します。懸念がある場合は、すぐに獣医師を通知します。</li>
	</ol>
</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+corneal+opacities+in+the+rat.">Induced corneal opacities in the rat.</a> Br J Ophthalmol. 51 (2), 124-129 (1967).</li></ol>

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この資料に示されている網膜注入法はインジェクター針が神経網膜を傷つけるまたは硝子体腔を乱すことがなく目の壁の外側の層 (強膜・脈絡膜・網膜色素複合体) を貫通強膜の経路です。代替トランス vitreal アプローチには、レンズの損傷を齧歯動物のレンズが硝子体腔の大半を占めているので、白内障につながるの潜在的なリスクがあります。このメソッドと比較して、本手法はリスクが...

人間の網膜は光感覚組織として目関数の後ろに位置し、視覚認知に重要な役割を果たしています。網膜細胞の機能不全や細胞死したがってビジョンの問題や永久的な盲目を引き起こします。変性や網膜の異なる層の細胞の機能不全を伴う疾患はその中で AMD は最も一般的なタイプとの先進国で高齢者に回復不能な失明の主要な原因、網膜変性疾患として知られています。1,2。AMD の病理学的プロセスが網膜色素上皮層と順番視細胞の生理学、神経網膜の萎縮およびビジョンの損失3につながるの RPE サポートを損なう基になるブルッフ膜の間「ドルーゼン」蓄積関連付け 4,5。これまでの治療がない高度な (非血管新生) AMD を乾燥します。幹細胞療法として再生医療の新しいパラダイムの出現は、健康な細胞の幹細胞の機能不全や死者の網膜色素上皮細胞を交換の希望をもたらします。確かに、広範な臨床研究移植の幹細胞 (例えば、ヒト胚性幹細胞)-RPE 変性動物モデルに派生の網膜色素上皮細胞が実行6、7にいくつかの進歩をされています。臨床試験8,9 (NCT01344993、ClinicalTrials.gov)。最近では、幹細胞が人間の網膜色素上皮層、人間の網膜色素上皮幹細胞 (hRPESCs) の居住者の代替ソース研究室によって識別され、現在 amd hRPESC 派生した網膜色素上皮細胞 (RPE-hRPESC) 移植療法の臨床研究で使用されて10,11,12,13。
網膜下注入の技術は、上記当社グループを含む複数のグループによる前臨床試験に適用されます。動物の網膜注入の 2 つの一般的なアプローチ: トランス vitreal と強膜。トランス vitreal 方法の利点はレンズに全体 vitreal 腔を越える前眼に浸透、浸透、subretinal に到達する目に後ろに網膜と針の端を直接観察することが外科医のスペース14,,1516。ただし、それは (前部と後部)、2 つの場所で網膜を中断すること、レンズを傷つけるリスクを運ぶし、針が取り消されるとき硝子体に細胞の逆流の可能性があります必要があります。対照的に、トランス強膜方法は、原則として、網膜と硝子体の関与を回避し、逆流が目を終了します。色素性の齧歯動物の外科医は当初、強膜の浸透を観察できるが、色素性の脈絡膜に通過後針の端が表示されなくなります。直接観察することがなく網膜に違反は一般的と網膜剥離、硝子体に細胞や血液の配信になります。また、眼の表面が湾曲しているために、強膜注射のため最も効果的などの針の角度と深さを知ることは非常に困難です。
この可視化の記事で光コヒーレンストモグラフィ (OCT)、注射部位の詳細な検査ができると手術後の評価を使用して通知強膜網膜注入法を紹介します。私たちの強膜注入法は、定義済みの場所、角度、および非常に低い外科的外傷と高信頼性を生成する注射針の深さを利用しています。ここでは、人間の AMD の前臨床モデル RCS ラットの網膜注入 hRPESC 網膜色素上皮細胞を具体的に示す.この注入法で正常にかつ一貫して救わ hRPESC 網膜色素上皮細胞に非常に高い成功率で RCS ラットの目の網膜。細胞の注射以前が認め RCS 視細胞の保全に注入13後少なくとも 2 ヶ月。この手順は、解剖顕微鏡の下で行われ、学ぶことは簡単です。(外科医と助手) 注射を実行する二人必要で、それぞれの動物の注入の平均時間は 5 分未満。定義された角度と注射針の深さは 10 月が正常網膜注入を達成するために利用可能な研究所を可能にします。それは再現性の高い網膜にアクセスできます、細胞移植のみならず、医薬品配送と遺伝子治療のために使用できます。

<table><tbody><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>25200-072</td><td></td></tr><tr><td>DNAse I</td><td>Sigma</td><td>DN-25</td><td></td></tr><tr><td>1xDulbecco&amp;rsquo;s Phosphate Buffered Saline without Calcium &amp;amp; Magnesium (1xDPBS-CMF)</td><td>Corning Cellgro</td><td>431219</td><td></td></tr><tr><td>Sterile Balanced Salt Solution (BSS)</td><td>Alcon</td><td>00065079550</td><td></td></tr><tr><td>Sterile eye wash</td><td>Moore Medical</td><td>75519</td><td></td></tr><tr><td>Sterile 0.9% saline</td><td>Hospira</td><td>488810</td><td></td></tr><tr><td>Proparacaine Hydrochloride Ophthalmic Solution (0.5%)</td><td>Akorn</td><td>17478026312</td><td></td></tr><tr><td>Tropicamide Ophthalmic Solution, USP (1%)</td><td>Bausch &amp;amp; Lomb</td><td>24208058559</td><td></td></tr><tr><td>Phenylepherine Ophtalmic Solution, USP (10%) stock</td><td>Bausch &amp;amp; Lomb</td><td>42702010305</td><td>This is used to make 2.5% Phenylepherine</td></tr><tr><td>Buprenex</td><td>Patterson</td><td>433502</td><td></td></tr><tr><td>Dexamethasone</td><td>APP Pharmaceuticals</td><td>63323051610</td><td></td></tr><tr><td>100% Ethanol</td><td>Thermo Scientific</td><td>615090040</td><td></td></tr><tr><td>70% Ethanol</td><td>Ricca Chemical Company</td><td>2546.70-5</td><td></td></tr><tr><td>Sterile GenTeal Lubricant Eye Gel</td><td>Novartis</td><td>78042947</td><td></td></tr><tr><td>Sterile Systane Ultra Lubricant Eye Drops</td><td>Alcon</td><td>00065143105</td><td></td></tr><tr><td>hRPESC-RPE cells</td><td>Not available commercially</td><td></td><td>Please refer to &quot;Reference #12&quot; for cell isolation and mainteinance.</td></tr><tr><td>24-well plates</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>Conical tubes (15 ml)</td><td>Sarstedt</td><td>62554002</td><td></td></tr><tr><td>Microcentrifuge cap with o-ring</td><td>LPS inc</td><td>L233126</td><td></td></tr><tr><td>Capless Microcentrifuge tubes (1.7 ml)</td><td>LPS inc</td><td>L233041</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>Sterile alcohol wipe</td><td>McKesson</td><td>58-204</td><td></td></tr><tr><td>Sterile cotton tip applicators</td><td>McKesson</td><td>24-106-2S</td><td></td></tr><tr><td>Sterile Weck-Cel spears</td><td>Beaver-Visitec International&amp;nbsp;</td><td>0008680</td><td></td></tr><tr><td>Sterile surgical drapes&amp;nbsp;</td><td>McKesson</td><td>25-515</td><td></td></tr><tr><td>Gauze</td><td>McKesson</td><td>16-4242</td><td></td></tr><tr><td>Nanofil syringe (10 ul)</td><td>World Precision Instruments</td><td>Nanofil</td><td></td></tr><tr><td>Nanofil beveled 33-gauge needle</td><td>World Precision Instruments</td><td>NF33BV-2</td><td></td></tr><tr><td>Insulin syringe needles 31-gauge</td><td>Becton Dickinson</td><td>328418</td><td></td></tr><tr><td>Rat toothed forceps</td><td>World Precision Instruments</td><td>555041FT</td><td></td></tr><tr><td>Vannas Micro Dissecting Spring Scissors</td><td>Roboz</td><td>RS-5602</td><td></td></tr><tr><td>Circulating water T pump&amp;nbsp;</td><td>Stryker</td><td>TP700</td><td></td></tr><tr><td>Heating pad</td><td>Kent Scientific</td><td>TPZ-814</td><td></td></tr><tr><td>Animal anesthesia system</td><td>World Precision Instruments</td><td>EZ-7000</td><td></td></tr><tr><td>Balance</td><td>Ohaus</td><td>PA1502</td><td></td></tr><tr><td>Stereo microscope</td><td>Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Microscope light source</td><td>Schott</td><td>ACE series</td><td></td></tr><tr><td>Bioptigen Envisu Spectral Domain Ophthalmic Imaging System</td><td>Bioptigen</td><td>R2210</td><td></td></tr><tr><td>Sterile black marker pen</td><td>Viscot Industries</td><td>1416S-100</td><td></td></tr><tr><td>Miniature measuring scale</td><td>Ted Pella Inc</td><td>13623</td><td></td></tr><tr><td>Infrared Basking Spot Lamp&amp;nbsp;</td><td>EXO-TERRA</td><td>PT2144</td><td>This is used as a heating lamp for animals during the post-surgical recovery&amp;nbsp; phase</td></tr></tbody></table>

development of a refined protocol for trans-scleral subretinal transplantation of human retinal pigment epithelial cells into rat eyes

加齢に伴う黄斑変性症 (AMD) などの網膜変性疾患は、世界的な不可逆的な視力喪失の主要な原因です。AMD は、機能的にサポートおよび解剖学的神経網膜の周りを囲むセルの単分子膜は、網膜色素上皮 (RPE) 細胞の退化によって特徴付けられます。非血管新生 AMD (AMD) の現在の薬理学的治療法は、病気の進行を遅くしか新規治療戦略の特定を目的とした研究を施行したビジョンを復元できません。変性の網膜色素上皮細胞を乾燥 AMD を将来的に治療するために健康な細胞の将来性に置き換えます。AMD のための幹細胞補充療法の広範な前臨床研究は、幹細胞由来網膜色素上皮細胞の網膜注入の技術を適用する動物モデルの網膜への移植を含みます。これらの前臨床動物研究で最も頻繁に使用されるアプローチは、針の端の直接可視化の欠乏によって困難になされる、網膜の損傷で起因できるしばしば強膜ルートを通じてです。針終了位置の直接観察が可能硝子体を通して別のアプローチがより目の組織が邪魔と手術のトラウマ高いリスクを運ぶ。我々 は正常にし一貫してラット網膜下腔網膜色素上皮細胞を提供し、過剰な網膜の損傷を避けるために定義された針の角度と深さを使用するリスクが少ないと再現性のある変更された強膜注入手法を開発しました。この方法で配信される細胞は、最低 2 ヶ月は王立外科医師 (RCS) ラットで結構効果があります以前実証されています。細胞移植のみならず小分子や遺伝子治療の配信、この手法を使用することができます。

この資料で説明する手法を使用すると、一貫して納品 hRPESC 網膜色素上皮細胞 RCS ラットの網膜下腔の場所、角度、および組織 (図 1 b-D にインジェクター針挿入の深さを正確に制御することにより).すぐに次の移植は、OCT 検査を行った注射部位及び移植の細胞によって作成された網膜下の水疱を観察します。手術後 10 月評価は注射や網膜の損?...

Watch this Scientific Journal Video about Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes at JoVE.com

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

網膜の注射は、加齢黄斑変性症に対する幹細胞補充療法の臨床研究で広く適用されています。この可視化の記事でラットの目に細胞を提供する強膜によるリスクの少ない、再現性と正確に変更された網膜注入手法について述べる。

強膜網膜移植ひと網膜色素上皮細胞のラットの目の洗練されたプロトコルの開発

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

development-refined-protocol-for-trans-scleral-subretinal

Research

JoVE Journal

Neuroscience

9.4K ビュー.  Neural Stem Cell Institute. 網膜の注射は、加齢黄斑変性症に対する幹細胞補充療法の臨床研究で広く適用されています。この可視化の記事でラットの目に細胞を提供する強膜によるリスクの少ない、再現性と正確に変更された網膜注入手法について述べる。

ビデオ: Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACSの精製された感光体前駆体細胞の成人マウス・レチナへの副腎移植

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

医学

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

懸濁液中の網膜色素上皮細胞を送達するげっ歯類における網膜下注射の実行

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

ウサギにおける網膜下手術のためのステッププロトコルによってステップ

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs.

ミニブタのキャリアへのRPEの網膜下移植:術前準備、手術手技、および術後ケアのガイドライン

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

生物工学

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

ネコ大型動物モデルにおけるヒト胚性幹細胞由来網膜組織の網膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

神経科学

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 ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

網膜変性マウスモデルにおける網膜下移植片の経瞳孔ガイド下経強膜移植

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

Development of a Surgical Technique for Subretinal Implants in Rats

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.

<meta charset="utf8"></head><body>ラットにおける網膜下インプラントの手術技術の開発

The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.

ラットにおける網膜下インプラントの手術技術の開発

Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been ...

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, ...

強膜網膜移植ひと網膜色素上皮細胞のラットの目の洗練されたプロトコルの開発

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ラットにおける網膜下インプラントの手術技術の開発

ラットにおける網膜下インプラントの手術技術の開発

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