Name: toxicity screens in human retinal organoids for pharmaceutical discovery
Uploaded: 2021-03-04T15:00:00
Description: Watch this Scientific Journal Video about Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery at JoVE.com

由洛伊医学研究所支持。我们要感谢Lowy家族对MacTel项目的支持。我们要感谢Mari Gantner，Mike Dorrell和Lea Scheppke的智力投入和协助准备手稿。

1. 解冻、传代和扩增 iPSC/ESC
注意：对于所有细胞培养步骤，请使用最佳实践来维持无菌细胞培养。
<ol><li>用基底膜基质培养基包覆 6 孔细胞培养板。<ol><li>要制备 1x 该培养基，请遵循产品规格或用 9 mL DMEM/F12 稀释 75 μL 冷基质培养基。在 6 孔板中每孔加入 1.5 mL 新鲜制备的 1x 培养基。在37°C孵育30分钟。</li>
		<li>吸出基底膜基质培养基，并用 3 mL DMEM/F12 冲洗每个孔。加入 2 mL ...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+retinal+development+and+disease+with+human+pluripotent+stem+cells.">Mimicking retinal development and disease with human pluripotent stem cells.</a> Investigative Ophthalmology and Visual Science. 57 (5), 1-9 (2016).</li><li>Gan, L., Cookson, M. R., Petrucelli, L., La Spada, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Converging+pathways+in+neurodegeneration,+from+genetics+to+mechanisms.">Converging pathways in neurodegeneration, from genetics to mechanisms.</a> Nature Neuroscience. 21 (10), 1300-1309 (2018).</li><li>Aung, K. Z., Wickremasinghe, S. S., Makeyeva, G., Robman, L., Guymer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+estimates+of+macular+telangiectasia+type+2:+the+Melbourne+collaborative+cohort+study.">The prevalence estimates of macular telangiectasia type 2: the Melbourne collaborative cohort study.</a> Retina. 30 (3), 473-478 (2010).</li><li>Chew, E. Y., 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=Effect+of+ciliary+neurotrophic+factor+on+retinal+neurodegeneration+in+patients+with+macular+telangiectasia+type+2:+A+randomized+clinical+trial.">Effect of ciliary neurotrophic factor on retinal neurodegeneration in patients with macular telangiectasia type 2: A randomized clinical trial.</a> Ophthalmology. 126 (4), 540-549 (2019).</li><li>Gass, J. D., Blodi, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+juxtafoveolar+retinal+telangiectasis.+Update+of+classification+and+follow-up+study.">Idiopathic juxtafoveolar retinal telangiectasis. Update of classification and follow-up study.</a> Ophthalmology. 100 (10), 1536-1546 (1993).</li><li>Klein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+macular+telangiectasia+type+2+in+the+Beaver+Dam+eye+study.">The prevalence of macular telangiectasia type 2 in the Beaver Dam eye study.</a> American Journal of Ophthalmology. 150 (1), 55-62 (2010).</li><li>Powner, M. B., 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=Loss+of+Muller's+cells+and+photoreceptors+in+macular+telangiectasia+type+2.">Loss of Muller's cells and photoreceptors in macular telangiectasia type 2.</a> Ophthalmology. 120 (11), 2344-2352 (2013).</li><li>Gantner, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine+and+lipid+metabolism+in+macular+disease+and+peripheral+neuropathy.">Serine and lipid metabolism in macular disease and peripheral neuropathy.</a> New England Journal of Medicine. 381 (15), 1422-1433 (2019).</li><li>Scerri, T. S., 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=Genome-wide+analyses+identify+common+variants+associated+with+macular+telangiectasia+type+2.">Genome-wide analyses identify common variants associated with macular telangiectasia type 2.</a> Nature Genetics. 49 (4), 559-567 (2017).</li><li>Alecu, I., 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=Cytotoxic+1-deoxysphingolipids+are+metabolized+by+a+cytochrome+P450-dependent+pathway.">Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway.</a> Journal of Lipid Research. 58 (1), 60-71 (2017).</li><li>Othman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fenofibrate+lowers+atypical+sphingolipids+in+plasma+of+dyslipidemic+patients:+A+novel+approach+for+treating+diabetic+neuropathy.">Fenofibrate lowers atypical sphingolipids in plasma of dyslipidemic patients: A novel approach for treating diabetic neuropathy.</a> Journal of Clinical Lipidology. 9 (4), 568-575 (2015).</li><li>Ohlemacher, S. K., Iglesias, C. L., Sridhar, A., Gamm, D. M., Meyer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+highly+enriched+populations+of+optic+vesicle-like+retinal+cells+from+human+pluripotent+stem+cells.">Generation of highly enriched populations of optic vesicle-like retinal cells from human pluripotent stem cells.</a> Curr Protoc Stem Cell Biol. 32, 1-20 (2015).</li><li>Zhong, X., 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=Generation+of+three-dimensional+retinal+tissue+with+functional+photoreceptors+from+human+iPSCs.">Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.</a> Nature Communication. 5, 4047 (2014).</li><li>Eiraku, M., 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472 (7341), 51-56 (2011).</li><li>Wahlin, K. J., 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=Photoreceptor+outer+segment-like+structures+in+long-term+3D+retinas+from+human+pluripotent+stem+cells.">Photoreceptor outer segment-like structures in long-term 3D retinas from human pluripotent stem cells.</a> Science Reports. 7 (1), 766 (2017).</li><li>Capowski, E. E., 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=Reproducibility+and+staging+of+3D+human+retinal+organoids+across+multiple+pluripotent+stem+cell+lines.">Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.</a> Development. 146 (1), (2019).</li><li>Luo, Z., 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=An+optimized+system+for+effective+derivation+of+three-dimensional+retinal+tissue+via+Wnt+signaling+regulation.">An optimized system for effective derivation of three-dimensional retinal tissue via Wnt signaling regulation.</a> Stem Cells. 36 (11), 1709-1722 (2018).</li><li>Ovando-Roche, P., 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=Use+of+bioreactors+for+culturing+human+retinal+organoids+improves+photoreceptor+yields.">Use of bioreactors for culturing human retinal organoids improves photoreceptor yields.</a> Stem Cell Research Therapy. 9 (1), 156 (2018).</li></ol>

鉴别方案差异 自从Yoshiki Sasai的20组发明自形成光学杯以来，许多实验室已经开发了产生视网膜类器官的方案，这些类器官几乎可以在每一步5，18，19，21中变化。详尽的协议列表可以在Capowski等人22中找到。我们在这里提供的分化方案是一种简单、低?...

在细胞培养和动物模型中对人类疾病进行建模为发现、修改和验证药物治疗提供了宝贵的工具，使他们能够从候选药物发展到批准的治疗。尽管体外和非人体体内模型的组合长期以来一直是药物开发管道的关键组成部分，但它们经常无法预测新型候选药物的临床性能1。显然需要开发技术，弥合简单的人类细胞单一培养和临床试验之间的差距。自组织三维组织培养物类器官的最新技术进步提高了它们对模型组织的保真度，使其成为临床前药物开发管道中的有前途的工具2。
与非人类体内模型相比，人类细胞培养的一个主要优势是能够复制人类新陈代谢的特定复杂性，即使在人类和小鼠等高阶脊椎动物之间也可能有很大差异3。然而，这种特异性可能会被组织复杂性的损失所掩盖;视网膜组织就是这种情况，其中多种细胞类型错综复杂地交织在一起，并且在细胞亚型之间具有独特的共生代谢相互作用，无法在单一培养中复制4。人类类器官提供了复杂人体组织的传真，具有细胞培养的可及性和可扩展性，有可能克服这些疾病建模平台的缺陷。
来自干细胞的视网膜类器官已被证明在模拟人类神经视网膜的复杂组织方面特别忠实5。这使得视网膜类器官模型成为研究和治疗视网膜疾病6，7的有前途的技术。迄今为止，视网膜类器官的大部分疾病建模都集中在单基因视网膜疾病上，其中视网膜类器官来源于具有致病遗传变异的iPSC系7。这些通常是高渗透性突变，表现为发育表型。在衰老疾病方面，基因突变和环境压力源影响正常发育的组织的有效工作较少。衰老的神经退行性疾病可能具有复杂的遗传和环境压力源的贡献，这些环境应激源本质上很难使用短期细胞培养物进行建模。然而，在许多情况下，这些复杂的疾病可以结合在共同的细胞或代谢应激源上，当在完全发育的人体组织上进行测试时，可以为衰老的神经退行性疾病提供强有力的见解8。
迟发性黄斑退行性疾病，黄斑毛细血管扩张II型（MacTel），是一种遗传性复杂的神经退行性疾病的一个很好的例子，它融合在一个常见的代谢缺陷上。MacTel 是一种罕见的视网膜衰老退行性疾病，导致黄斑中光感受器和穆勒胶质细胞丢失，导致中心视力进行性丧失9，10，11，12，13。在MacTel中，一种未确定的，可能是多因素的遗传导致患者循环丝氨酸的共同减少，导致称为脱氧鞘脂（deoxySL）的神经毒性脂质种类增加14，15。为了证明脱氧SL的积累对视网膜有毒并验证潜在的药物治疗，我们开发了该协议来测定人视网膜类器官中的光感受器毒性14。
在这里，我们概述了用于区分人视网膜类器官，使用类器官建立毒性和救援测定以及量化结果的特定方案。我们提供了一个成功的例子，我们确定了可疑致病剂deoxySL的组织特异性毒性，并验证了使用安全的仿制药非诺贝特来治疗脱氧SL诱导的视网膜毒性。先前的工作表明，非诺贝特可以增加患者脱氧SL的降解并降低循环脱氧SL，然而，其在降低脱氧SL诱导的视网膜毒性方面的功效尚未经过测试16，17。尽管我们提出了一个具体的例子，但该协议可用于评估任何数量的代谢/环境应激源和潜在治疗药物对视网膜组织的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>125mL Erlenmeyer Flasks</td>
			<td>VWR</td>
			<td>89095-258</td>
			<td></td>
		</tr>
		<tr>
			<td>1-deoxysphinganine</td>
			<td>Avanti</td>
			<td>860493</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement, minus vitamin A</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaver 6900 Mini-Blade</td>
			<td>Beaver-Visitec</td>
			<td>BEAVER6900</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Sucrose</td>
			<td>VWR</td>
			<td>97061-432</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo-fisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II, powder</td>
			<td>Gibco</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Gibco</td>
			<td>11995073</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Ig-G, Alexa Fluor plus 555</td>
			<td>Thermo-fisher</td>
			<td>A32794</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey serum</td>
			<td>Sigma</td>
			<td>D9663-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, Heat Inactivated</td>
			<td>Corning</td>
			<td>45001-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Fenofibrate</td>
			<td>Sigma</td>
			<td>F6020</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Stemcell Technologies</td>
			<td>7980</td>
			<td></td>
		</tr>
		<tr>
			<td>In Situ Cell Death Detection Kit, Fluorescin</td>
			<td>Sigma</td>
			<td>11684795910</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, growth factor reduced</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR 1</td>
			<td>Stemcell Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 16% Formaldehyde</td>
			<td>Thermo-fisher</td>
			<td>28906</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-Recoverin antibody</td>
			<td>Millipore</td>
			<td>AB5585</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip Sterile Disposable Vacuum Filter Units</td>
			<td>MilliporeSigma</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Plus- O.C.T. compound</td>
			<td>Fisher Scientific</td>
			<td>23-730-571</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Cryomold</td>
			<td>EMS</td>
			<td>62534-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment 6 well Plates</td>
			<td>Corning</td>
			<td>29443-030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment 75cm2 U-Flask</td>
			<td>Corning</td>
			<td>3814</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Filtration System</td>
			<td>VWR</td>
			<td>10040-436</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield-mounting medium</td>
			<td>vector Labs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>wax pen-ImmEdge</td>
			<td>vector Labs</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 Dihydrochloride (Rock inhibitor)</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td></td>
		</tr>
	</tbody>
</table>

toxicity screens in human retinal organoids for pharmaceutical discovery

类器官为研究疾病机制和治疗提供了一个有前途的平台，直接在具有细胞培养多功能性和通量的人体组织背景下。成熟的人视网膜类器官用于筛选与年龄相关的视网膜退行性疾病黄斑毛细血管扩张症 2 型 （MacTel） 的潜在药物治疗。
我们最近表明，MacTel 可能是由非典型脂质物种脱氧鞘脂 （deoxySL） 水平升高引起的。这些脂质对视网膜有毒，并可能导致MacTel患者发生的感光器损失。为了筛选药物预防脱氧SL感光器毒性的能力，我们从非MacTel诱导的多能干细胞（iPSC）系生成了人视网膜类器官，并将它们成熟到有丝分裂后年龄，在那里它们发育出视网膜的所有神经元谱系衍生细胞，包括功能成熟的光感受器。用脱氧SL代谢物处理视网膜类器官，并使用免疫组织化学测量光感受器层内的细胞凋亡。使用该毒性模型，筛选了防止脱氧SL诱导的感光器死亡的药理化合物。使用靶向候选方法，我们确定非诺贝特（一种通常用于治疗高胆固醇和甘油三酯的药物）也可以预防视网膜细胞中的脱氧SL毒性。
毒性筛查成功鉴定出FDA批准的可以预防感光器死亡的药物。由于测试了高度相关的疾病模型，这是一个直接可操作的发现。该平台可以轻松修改，以测试任意数量的代谢应激源和潜在的药物干预，以用于视网膜疾病的未来治疗发现。

视网膜类器官由非MacTel对照iPSC系产生。类器官在培养中达到26周后，将它们选中并分成实验组。用不同浓度的脱氧SA处理类器官，以确定脱氧SA是否对光感受器有毒。测试四种浓度的脱氧SA，从0到1μM（图2），类器官处理8天，每隔一天更换一次培养基。响应脱氧SA的细胞死亡是浓度依赖性的，可在低至50nM脱氧SA中检测到（图2D）。测试的最高浓度1μM脱氧S...

Watch this Scientific Journal Video about Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery at JoVE.com

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

在这里，我们提出了一个分步方案来生成成熟的人视网膜类器官，并在光感受器毒性测定中利用它们来确定年龄相关性视网膜退行性疾病黄斑毛细血管扩张症 2 型 （MacTel） 的候选药物。

用于药物发现的人视网膜类器官毒性筛选

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

该协议提供了一种临床前方法，以发现和验证直接在人类视网膜组织内代谢性视网膜疾病的药物治疗。该技术将人体组织中代谢的复杂性与细胞培养的多功能性和通量相结合。我们已经使用该测定法获取治疗视网膜退行性疾病MacTel的临床前数据，使用一种常见且安全的脂质改变药物非诺贝特，该药物目前正在进行临床安全性试验。这种方法可以应用于导致疾病的任意数量的常见视网膜应激源，包括营养缺乏、缺氧、光毒性和其他有毒脂质。在培养的第 24 周，检查类器官是否在类器官外部形成基本片段。到第26周到第28周，外段应该已经形成了厚厚的一层。当可以观察到增厚的怀疑者段时，将一毫摩尔脱氧SA储备溶液稀释至0.51。在三毫升视网膜分化培养基中加入一微摩尔和两微摩尔浓度加。使用解剖显微镜和无菌探针，选择四组大小和形状大致相同的类器官，每组至少五个类器官，并将这些组分成超低附着 6 孔板的单个孔。收集完所有类器官后，从每个孔中去除尽可能多的培养基，并向每个孔中加入一种浓度的脱氧SA溶液。将载体对照添加到第四组类器官中。培养两天后，用适当的新鲜脱氧SA或对照稀释液替换培养物上清液。培养四天后，嵌入冷冻切片并根据标准方案对类器官进行染色，以评估每种培养物中的细胞死亡。染色后，使用5至10倍放大倍率定位切片类器官切片中完整，结构良好的区域，并在对类器官的代表性横截面进行采样的平面中。该切片应具有至少几个细胞厚的光感受器的独特外核层，以及没有恢复和染色的独立且独特的细胞核层。构图并将放大倍率增加到 20 倍。然后，再次构图幻灯片，用尽可能多的感光层填充图像。设置 Z 平面并设置 Z 范围的上限下限，以对切片的整个深度进行成像。然后在Z-stack采集中对荧光团的所有三个通道进行成像，并将图像保存为保留每像素面积比的文件格式。要量化每个样品中的死细胞数量，请在斐济打开图像堆栈。在生物格式的导入选项窗口中，确认未选中拆分为单独窗口部分下的框，然后选择图像、堆栈和 Z 项目以创建新图像进行量化。选择显示恢复和染色的图像通道，并使用多边形选择工具勾勒出图像中感光器的连续区域。单击分析和设置测量值，然后选择面积框。单击分析和测量，以测量光感受器的面积，以计数垂死的细胞。测量结果将显示在弹出窗口中。要计数所选感光器区域中的细胞凋亡阳性细胞核，请右键单击点工具以选择多点工具，然后选择与DAPI阳性细胞核重叠的细胞凋亡阳性染色并恢复和染色。在通道之间切换以验证阳性细胞。然后将细胞凋亡阳性细胞的数量除以面积，以获得每个类器官光感受器内细胞死亡的标准化值。确定诱导靶细胞死亡的脱氧SA浓度后，使用无菌探针选择三组大小和形状大致相同的类器官，每组至少五个类器官。并将这些组添加到超低附着6孔板的各个孔中。将目标细胞死亡浓度的脱氧SA、补充有目标药物或对照培养基的脱氧SA的靶细胞死亡浓度加入到适当的孔或类器官中。两天后，用适当浓度的新鲜处理或对照培养基替换每个孔中的上清液。培养四天后，如图所示评估培养物的细胞死亡情况。在这项代表性分析中，用四种浓度的脱氧SA处理由非黄斑毛细血管扩张2型控制诱导的多能干细胞系产生的视网膜类器官。响应脱氧SA的细胞死亡是浓度依赖性的，并且可以在低至50纳摩尔的脱氧SA中检测到。测试的最高浓度诱导了强大的细胞死亡，同时仍然保持视网膜类器官的完整性。用20微摩尔非诺贝特处理可防止脱氧SA诱导的视网膜类器官光受体毒性，治疗4天后显着减少约80%的细胞死亡。请记住选择形态良好且健康的类器官，以确保在视网膜细胞中进行测定。可以处理类器官以进行RNA收集，以量化基因表达，从而可以评估应激信号并验证毒性作用和救援。使用这种技术，我们已经能够测定类器官中多种不同脂质的下游代谢，开辟了人类视网膜中脂质代谢的研究。

Research

JoVE Journal

Bioengineering

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

GENPLAT：生物质酶的发现和鸡尾酒优化的自动化平台

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

路西法黄 - 人类血脑屏障细胞模型中一个健壮的细胞渗透标记

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...