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>

<ol>
	<li>Murdaugh, L. S., Wang, Z., Del Priore, L. V., Dillon, J., Gaillard, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+accumulation+of+3-nitrotyrosine+and+nitro-A2E+in+human+Bruch's+membrane.">Age-related accumulation of 3-nitrotyrosine and nitro-A2E in human Bruch's membrane.</a> Exp. Eye Res. 90 (5), 564-571 (2010).</li><li>Wiradjaja, F., DiTommaso, T., Smyth, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membranes+in+development+and+disease.">Basement membranes in development and disease.</a> Birth Defects Res C Embryo Today. 90 (1), 8-31 (2010).</li><li>Nystrom, A., Bornert, O., Kuhl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+for+basement+membrane-linked+diseases.">Cell therapy for basement membrane-linked diseases.</a> Matrix Biol. , (2016).</li><li>Pauleikhoff, D., Harper, C. A., Marshall, J., Bird, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+changes+in+Bruch's+membrane.+A+histochemical+and+morphologic+study.">Aging changes in Bruch's membrane. A histochemical and morphologic study.</a> Ophthalmology. 97 (2), 171-178 (1990).</li><li>Marshall, G. E., Konstas, A. G., Reid, G. G., Edwards, J. G., Lee, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+collagen+and+laminin+in+Bruch's+membrane+and+basal+linear+deposit+in+the+human+macula.">Type IV collagen and laminin in Bruch's membrane and basal linear deposit in the human macula.</a> Brit. J. Ophthalmol. 76 (10), 607-614 (1992).</li><li>Sarks, S. H., Arnold, J. J., Killingsworth, M. C., Sarks, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+drusen+formation+in+the+normal+and+aging+eye+and+their+relation+to+age+related+maculopathy:+a+clinicopathological+study.">Early drusen formation in the normal and aging eye and their relation to age related maculopathy: a clinicopathological study.</a> Brit. J. Ophthalmol. 83 (3), 358-368 (1999).</li><li>Spraul, C. W., Lang, G. E., Grossniklaus, H. E., Lang, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+and+morphometric+analysis+of+the+choroid,+Bruch's+membrane,+and+retinal+pigment+epithelium+in+postmortem+eyes+with+age-related+macular+degeneration+and+histologic+examination+of+surgically+excised+choroidal+neovascular+membranes.">Histologic and morphometric analysis of the choroid, Bruch's membrane, and retinal pigment epithelium in postmortem eyes with age-related macular degeneration and histologic examination of surgically excised choroidal neovascular membranes.</a> Surv. Ophthalmol. 44 (Suppl 1), S10-S32 (1999).</li><li>Abdelsalam, A., Del Priore, L., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+in+age-related+macular+degeneration:+pathogenesis,+natural+course,+and+laser+photocoagulation-induced+regression.">Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation-induced regression.</a> Surv. Ophthalmol. 44 (1), 1-29 (1999).</li><li>Mullins, R. F., Aptsiauri, N., Hageman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+composition+of+drusen+associated+with+glomerulonephritis:+implications+for+the+role+of+complement+activation+in+drusen+biogenesis.">Structure and composition of drusen associated with glomerulonephritis: implications for the role of complement activation in drusen biogenesis.</a> Eye. 15 (Pt 3), 390-395 (2001).</li><li>Caldwell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+alterations+precede+vascularization+of+the+retinal+pigment+epithelium+in+dystrophic+rats.">Extracellular matrix alterations precede vascularization of the retinal pigment epithelium in dystrophic rats.</a> Curr. Eye Res. 8 (9), 907-921 (1989).</li><li>Pauleikhoff, D., Harper, C. A., Marshall, J., Bird, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+changes+in+Bruch's+membrane.+A+histochemical+and+morphologic+study.">Aging changes in Bruch's membrane. A histochemical and morphologic study.</a> Ophthalmology. 97 (2), 171-178 (1990).</li><li>Abdelsalam, A., Del Priore, L., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+in+age-related+macular+degeneration:+pathogenesis,+natural+course,+and+laser+photocoagulation-induced+regression.">Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation-induced regression.</a> Surv. Ophthalmol. 44 (1), 1-29 (1999).</li><li>Spraul, C. W., Lang, G. E., Grossniklaus, H. E., Lang, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+and+morphometric+analysis+of+the+choroid,+Bruch's+membrane,+and+retinal+pigment+epithelium+in+postmortem+eyes+with+age-related+macular+degeneration+and+histologic+examination+of+surgically+excised+choroidal+neovascular+membranes.">Histologic and morphometric analysis of the choroid, Bruch's membrane, and retinal pigment epithelium in postmortem eyes with age-related macular degeneration and histologic examination of surgically excised choroidal neovascular membranes.</a> Surv Ophthalmol. 44, S10-S32 (1999).</li><li>Del Priore, L. V., Tezel, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reattachment+rate+of+human+retinal+pigment+epithelium+to+layers+of+human+Bruch's+membrane.">Reattachment rate of human retinal pigment epithelium to layers of human Bruch's membrane.</a> Arch. Ophthalmol. 116 (3), 335-341 (1998).</li><li>Ho, T. C., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reattachment+of+cultured+human+retinal+pigment+epithelium+to+extracellular+matrix+and+human+Bruch's+membrane.">Reattachment of cultured human retinal pigment epithelium to extracellular matrix and human Bruch's membrane.</a> Invest. Ophth. Vis. Sci. 38 (6), 1110-1118 (1997).</li><li>Tezel, T. H., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reattachment+to+a+substrate+prevents+apoptosis+of+human+retinal+pigment+epithelium.">Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium.</a> Graefes Arch. Clin. Exp. Ophthalmol. 235 (1), 41-47 (1997).</li><li>Tezel, T. H., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF+beta+secretion+modulates+the+density-dependent+growth+of+pig+retinal+pigment+epithelium+in+vitro.">TGF beta secretion modulates the density-dependent growth of pig retinal pigment epithelium in vitro.</a> Ophthalmic Res. 31 (3), 192-202 (1999).</li><li>Gullapalli, V. K., Sugino, I. K., Van Patten, Y., Shah, S., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+RPE+survival+on+aged+submacular+human+Bruch's+membrane.">Impaired RPE survival on aged submacular human Bruch's membrane.</a> Exp. Eye Res. 80 (2), 235-248 (2005).</li><li>Wang, H., Yagi, F., Cheewatrakoolpong, N., Sugino, I. K., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+study+of+retinal+pigment+epithelium+sheet+transplants+onto+Bruch's+membrane.">Short-term study of retinal pigment epithelium sheet transplants onto Bruch's membrane.</a> Exp. Eye Res. 78 (1), 53-65 (2004).</li><li>Tezel, T. H., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repopulation+of+different+layers+of+host+human+Bruch's+membrane+by+retinal+pigment+epithelial+cell+grafts.">Repopulation of different layers of host human Bruch's membrane by retinal pigment epithelial cell grafts.</a> Invest. Ophth. Vis. Sci. 40 (3), 767-774 (1999).</li><li>Tezel, T. H., Del Priore, L. V., Kaplan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reengineering+of+aged+Bruch's+membrane+to+enhance+retinal+pigment+epithelium+repopulation.">Reengineering of aged Bruch's membrane to enhance retinal pigment epithelium repopulation.</a> Invest. Ophth. Vis. Sci. 45 (9), 3337-3348 (2004).</li><li>Castellarin, A. A., Sugino, I. K., Vargas, J. A., Parolini, B., Lui, G. M., Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transplantation+of+fetal+human+retinal+pigment+epithelial+cells+onto+human+cadaver+Bruch's+membrane.">In vitro transplantation of fetal human retinal pigment epithelial cells onto human cadaver Bruch's membrane.</a> Exp. Eye Res. 66 (1), 49-67 (1998).</li><li>Nguyen, H. V., Li, Y., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+iPSC-derived+RPE+for+modeling+of+retinal+diseases.">Patient-specific iPSC-derived RPE for modeling of retinal diseases.</a> J. Clin. Med. 4 (4), 567-578 (2015).</li><li>Fletcher, E. L., Jobling, A. I., Greferath, U., Mills, S. A., Waugh, M., Ho, T., De Longh, R. U., Phipps, J. A., Vessey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+age-related+macular+degeneration+using+animal+models.">Studying age-related macular degeneration using animal models.</a> Optometry Vision Sci. 91 (8), 878-886 (2014).</li><li>Del Priore, L. V., Tezel, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reattachment+rate+of+human+retinal+pigment+epithelium+to+layers+of+human+Bruch's+membrane.">Reattachment rate of human retinal pigment epithelium to layers of human Bruch's membrane.</a> Arch. Ophthalmol. 116 (3), 335-341 (1998).</li><li>Tezel, T. H., Kaplan, H. J., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+human+retinal+pigment+epithelial+cells+seeded+onto+layers+of+human+Bruch's+membrane.">Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch's membrane.</a> Invest. Ophth. Vis. Sci. 40 (2), 467-476 (1999).</li><li>Cai, H., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruch+membrane+aging+alters+the+gene+expression+profile+of+human+retinal+pigment+epithelium.">Bruch membrane aging alters the gene expression profile of human retinal pigment epithelium.</a> Curr. Eye Res. 31 (2), 181-189 (2006).</li><li>Moreira, E. F., Cai, H., Tezel, T. H., Fields, M. A., Del Priore, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reengineering+human+Bruch's+membrane+increases+rod+outer+segment+phagocytosis+by+human+retinal+pigment+epithelium.">Reengineering human Bruch's membrane increases rod outer segment phagocytosis by human retinal pigment epithelium.</a> Transl. Vis. Sci. Technol. 4 (5), 10 (2015).</li><li>Sun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruch's+membrane+aging+decreases+phagocytosis+of+outer+segments+by+retinal+pigment+epithelium.">Bruch's membrane aging decreases phagocytosis of outer segments by retinal pigment epithelium.</a> Mol. Vis. 13, 2310-2319 (2007).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1219">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:599px;">Human donor globes</td>
			<td style="width:305px;">National Disease Research Interchange, NDRI</td>
			<td style="width:148px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbon-dioxide independent media</td>
			<td>Thermo Fisher Scientific</td>
			<td>18045-088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mm polystyrene petri dish</td>
			<td>Corning Inc.</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s phosphate buffered saline, PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrophobic 65 &#181;m-thick polytetrafluoroethylene membrane with 0.2 &#181;m pores</td>
			<td>Merck Millipore</td>
			<td>JGWP04700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand glass microanalysis vacuum filter holder system&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>09-753-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A2576-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 &#215; 10mm culture dish</td>
			<td>VWR International</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trephine</td>
			<td>Accutome</td>
			<td>AM0570 60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well plate</td>
			<td>Corning Inc.</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum essential media (MEM)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11095-080&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin G</td>
			<td>Sigma-Aldrich</td>
			<td>P3032-1MU</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>1397-89-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium hydroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>1336-21-6 
			&#160;</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/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/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/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

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

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

最適化された<em&gt;元OVO</em&gt;開発の進んだ段階にニワトリ胚の培養

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

発生生物学

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

幹細胞から網膜色素上皮細胞の効率的な導出

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

胚様体の異なるサイズによって人工多能性幹（iPS）細胞から網膜色素上皮（RPE）の導出

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

臨床的に関連の確立<em&gt;エクスビボ</emネイティブ微小環境における上皮創傷修復を調査するための&gt;モック白内障手術モデル

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Basicおよび前臨床研究のための密閉型細胞培養系でヒト多能性および神経幹細胞を培養します

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

アン<em&gt; ex vivoで</em&gt;メソッド培養ラット腸間膜微小血管ネットワークのタイムラプスイメージングのための

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

生体内でひと脂肪由来間葉系幹細胞が脂溶性膜の蛍光色素をラット膝関節変形性関節症モデルでの追跡

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

ヒト胚性幹細胞に由来する移植適切な網膜色素上皮組織をエンジニア リング

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

効率的かつスケーラブルな臨床的に互換性のある角膜輪部上皮幹細胞ひと多能性幹細胞からの分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...