我们感谢美国国家眼科研究所（NEI）组织学核心使用蔡司共聚焦系统。这项工作得到了NEI IRP基金的支持（授权号ZIA EY000533-04）。

所有方案步骤均遵循NIH人类研究伦理委员会制定的指导方针。根据美国政府的45 CFR 46指南，干细胞工作和患者样本采集已获得NIH人类研究保护办公室（OHRP）下属的联合神经科学机构审查委员会（CNS IRB）的批准。根据赫尔辛基宣言规定的标准，使用CNS IRB批准的同意书收集患者样本，协议号为NCT01432847（https://clinicaltrials.gov/ct2/show/NCT01432847?cond=NCT01432847&draw=2&rank=1）。
1. iRPE 生成
<ol><li>按照 Sharma 等人发表的方案从患者血液来源的 iPSC 中生成 iRPE，202211 （图 2 和 图 3）。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65503/65503fig02v2.jpg"> 图 2:iRPE 分化和成熟的示意图。 为了产生iRPE，遵循既定的分化方案，并使细胞成熟5周。由此产生的细胞培养物充当体 外 模型，可以通过各种治疗方法进行操作，以模拟AMD和Stargardt病等疾病的RPE功能障碍。 <a href="https://www.jove.com/files/ftp_upload/65503/65503fig02largev2.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65503/65503fig03.jpg"> 图 3:成功和不成功的 RPE 分化和成熟的代表性图像。 在 iRPE 方案的第 42 天显示了 TJP1 RPE 10 倍放大倍率下的两个明场图像。（A）成功的分化和成熟将显示出与色素沉着和多边形形态的融合RPE。（B）不成功的分化和成熟将显示死亡细胞的簇，如图所示。 <a href="https://www.jove.com/files/ftp_upload/65503/65503fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
2. RPE 维护培养基 （RPE-MM） 制备
<ol><li>将N 2补充剂在4°C下解冻过夜。在室温（RT）下解冻所有其他试剂。</li>
	<li>在无菌条件下，按照Sharma等人制定的方案，按照Sharma等人制定的方案，以列出的稀释因子加入 表1 中列出的试剂111。</li>
	<li>充分混合培养基并使用0.22μm过滤装置进行过滤。 注意:如果储存在4°C，培养基适合在2周内使用。</li>
</ol>3. 96孔板播种
<ol><li>在室温下解冻等分试样玻连蛋白3-5分钟或直到冰完全融化。</li>
	<li>用1x Dulbecco的磷酸盐缓冲盐水（DPBS）稀释玻连蛋白，以使用1:200稀释度（玻连蛋白:DPBS）获得所需的工作溶液。对于 96 孔板，在每个孔中涂上 200 μL 工作溶液。</li>
	<li>将解冻的ROCK抑制剂（Y-27632二盐酸盐）与RPE-MM以1:1000稀释度混合，以达到10μM的最终浓度。这是RPE细胞的电镀介质。</li>
	<li>使用自动细胞解冻系统解冻iRPE小瓶，并将iRPE细胞悬液转移到50 mL管中。</li>
	<li>用电镀介质以1:10稀释度稀释细胞悬液。将试管以400× g 离心5分钟。</li>
	<li>小心吸出上清液并将细胞重悬于10mL电镀介质中。</li>
	<li>将 400 μL 电镀介质与 100 μL 重悬细胞溶液混合以进行细胞计数。使用该等分试样使用细胞活力计数器确定细胞悬液的活细胞浓度。</li>
	<li>用电镀培养基稀释细胞悬液至终浓度为60，000个细胞/ mL。</li>
	<li>从 96 孔板中完全吸出玻连蛋白包衣溶液，并将 200 μL 细胞悬液分配到每个孔中。将有大约 12，000 个细胞/孔或 ~200 个细胞/mm2。</li>
	<li>将接种的细胞板在37°C和5%CO2下孵育48小时。48小时后，将培养基更换为RPE-MM，无需补充ROCK抑制剂。在 5 周的成熟期内，每 2-3 天更换一次培养基。</li>
</ol>4. 体外 疾病模型
<ol><li>补体感受态人血清 （CC-HS） 治疗<ol><li>将人补体感受态血清在 4°C 下解冻过夜。</li>
		<li>制备 CC-HS 并补体无功能人血清 （CI-HS） 培养基。<ol><li>要制备 5% CC-HS 培养基，将解冻的补体感受态人血清与 RPE-MM 以 1:20 稀释度混合。使用前通过0.22μm介质过滤器过滤溶液。</li>
			<li>为了制备5%补体无功能人血清（CI-HS）培养基，首先在57°C水浴中加热灭活CC-HS30分钟，然后以1:20稀释度与培养基混合。使用前通过0.22μm介质过滤器过滤溶液。</li>
		</ol></li>
		<li>血清用200μL5%CC-HS或5%CI-HS培养基处理细胞，总孵育时间为48小时，24小时后刷新培养基。</li>
		<li>用1x DPBS洗涤细胞，并在室温下用4%多聚甲醛固定20分钟。 再次用1x DPBS洗涤并将样品储存在4°C，浸入200μLDPBS中。</li>
		<li>可选:如果需要，从平板中裂解细胞以仅显示亚RPE脂质沉积。<ol><li>要裂解细胞并仅留下脂质沉积物，请取出培养基并向每个孔中加入 200 μL 去离子水。</li>
			<li>孵育10-15分钟，上下移液直至细胞被移除。用 200 μL 去离子水再次洗涤，并立即用 4% 多聚甲醛固定细胞。</li>
			<li>使用Hoechst通过核染色确认细胞去除功效。以 1:2000 稀释度将 Hoechst 加入含有 1% 牛血清白蛋白 （BSA）、0.5% 吐温 20 和 0.5% Triton-X 100 的 1x DPBS 溶液中。在室温下在黑暗中孵育1小时。随后，用 1x DPBS 洗涤。</li>
		</ol></li>
	</ol></li>
	<li>iRPE的感光器外段（POS）治疗<ol><li>收银机准备<ol><li>从-80°C储存中取出POS颗粒管，并在4°C的有盖冰桶中解冻过夜。</li>
			<li>通过将 10 g 蔗糖与 40 mL 双去离子 H 2 O （ddH2O） 混合来制备 POS 洗涤缓冲液。</li>
			<li>在40-50°C下加热混合物，同时轻轻搅拌15分钟。向混合物中加入840mg碳酸氢钠，并在加热10分钟时搅拌。</li>
			<li>根据需要，用ddH2O将POS洗涤缓冲液的总体积调节至100mL，并根据需要用1N HCl或1N NaOH将溶液的pH调节至8.3。使用0.22μm过滤器过滤洗涤溶液。 注意:协议可以在此处暂停;POS洗涤缓冲液可以在4°C下储存过夜。</li>
			<li>解冻后，将沉淀悬浮在 15 mL POS 洗涤缓冲液中。在颗粒悬浮期间要轻柔，以确保 POS 的完整性。将POS悬浮液在4°C下以600× g 离心20分钟，然后吸出上清液。</li>
			<li>将 POS 沉淀重悬于 10 mL POS 洗涤缓冲液中。</li>
			<li>取出 100 μL 等分试样的 POS + POS 洗涤缓冲液（POS 溶液），并在 400 μL 1x DPBS 中稀释。将 50 μL 稀释的 POS 溶液铺在血琼脂平板和琼脂糖平板上，以检查细菌和真菌污染物。为每个准备阳性对照，并在37°C下孵育所有板48小时。</li>
			<li>通过在检测孔中加入 1 μL POS 溶液来检测支原体，从而进行 qPCR 测定。要扩增 DNA 片段，请执行 40 个变性循环（95°C，15 秒）以及退火和伸长循环（60°C，1 分钟）。用于检测POS样品中支原体的正向和反向引物如下: 前向入门: GGA TTA GAT ACC CTG GTA GTC CAC G 反向引物:CGT CAA TTC CTT TAA GTT TCA CTC TTG GC</li>
			<li>使用细胞分析仪测量POS浓度，并根据需要等分试样。对于一个带有RPE细胞的96孔板的孔，3 x 106 POS就足够了。所需比率为 10 个 POS/RPE 单元。将等分试样储存在80°C以备将来使用。</li>
		</ol></li>
		<li>将 POS 添加到单元格中<ol><li>在冰浴中解冻POS小瓶。</li>
			<li>将计算的制备量的POS与RPE-MM混合，并每天用POS处理细胞一次，持续7天。 注意:每天准备新鲜的POS解决方案。</li>
			<li>用1x DPBS洗涤细胞，然后在室温下用4%多聚甲醛固定20分钟。 再次用DPBS洗涤并将样品储存在4°C，浸入200μLDPBS中。</li>
		</ol></li>
	</ol></li>
</ol>5. 亚 RPE 沉积物的染色
<ol><li>尼罗河红染色方案<ol><li>PFA固定后，用1x DPBS洗涤样品3次。 注意:如果不立即使用，可以在此处暂停协议，但样品必须储存在4°C的1x DPBS + 0.02%叠氮化钠溶液中。</li>
		<li>要制备尼罗河红储备溶液，请将尼罗红粉末溶解在丙酮中，浓度为 3 mg/mL。定期混合在室温下孵育15分钟。用0.22μm过滤器过滤溶液一次或两次，具体取决于溶液中残留的沉淀物水平。 注意:保护储备溶液避光。</li>
		<li>为了制备工作溶液，在1x DPBS中以1:500的比例稀释储备溶液。在摇床上向样品中加入 200 μL 工作溶液 30 分钟，并保护其免受光照。</li>
		<li>用1x PBS洗涤3次，并将样品储存在4°C，浸入200μLDPBS中。 注意:如果在Transwell而不是96孔板上进行实验，则可以将样品安装在带有安装介质的载玻片上，用玻璃盖玻片覆盖，并用透明指甲油密封。应注意将样品朝上安装。</li>
	</ol></li>
	<li>BODIPY染色方案<ol><li>对于储备溶液，将BODIPY溶解在无水二甲基亚砜（DMSO）中，以达到3.8mM的储备浓度。</li>
		<li>对于PFA固定样品，在1x DPBS中以1:300的比例稀释BODIPY原液。向细胞中加入 200 μL，并在室温下在摇杆上孵育过夜。</li>
		<li>用1x DPBS洗涤3次，并将样品储存在4°C，浸入200μLDPBS中。 注意:如果在Transwell而不是96孔板上进行实验，则可以将样品安装在带有安装介质的载玻片上，用玻璃盖玻片覆盖，并用透明指甲油密封。应注意将样品朝上安装。</li>
	</ol></li>
	<li>APOE 免疫染色方案<ol><li>将 1x DPBS 与 1% 牛血清白蛋白 （BSA）、0.5% 吐温 20 和 0.5% Triton-X 100 混合，制成缓冲溶液。</li>
		<li>对于PFA固定样品，在室温下将样品在200μL缓冲溶液中封闭并透化1小时。</li>
		<li>在缓冲溶液中加入以1:100稀释的APOE一抗，并在室温下孵育过夜。</li>
		<li>第二天，用1x DPBS洗涤样品3次。</li>
		<li>在缓冲溶液中以1:1000稀释度加入二抗，并在室温下向细胞中加入200μL溶液1小时。</li>
		<li>用1x DPBS洗涤3次，并将样品储存在4°C，浸入200μLDPBS中。 注意:如果在Transwell而不是96孔板上进行实验，则可以将样品安装在带有安装介质（Fluoromount）的载玻片上，用玻璃盖玻片覆盖，并用透明指甲油密封。应注意将样品朝上安装。</li>
	</ol></li>
</ol>6. 图像自动化和处理
<ol><li>自动图像扫描 注:本研究使用了蔡司LSM 800倒置共聚焦扫描显微镜和ZEN 3.2（蓝色版）软件。确保在成像前将96孔板加热至室温至少60分钟，以避免在扫描过程中由于介质折射率随温度变化而变化而导致焦平面漂移。<ol><li>使用共聚焦显微镜和40倍物镜，使用适当的荧光通道为所使用的脂质标志物和任何其他抗体创建扫描图谱。</li>
		<li>使用 "磁贴 "复选框设置图像自动化。要校准 96 孔板，请确保输入并选择正确的样品载体测量值。然后，单击 校准 按钮根据说明校准板，这需要使用10倍物镜。</li>
		<li>选择"高级设置"视图以选择合适的孔，并使用"位置"功能在孔中心附近添加3个不同的成像点。这可以在"位置"子选项卡下手动完成，也可以使用"位置设置"选项卡并选择"按承运人设置"随机完成。对相同染色的所有孔重复此操作。</li>
		<li>为了在自动化过程中获得最佳对焦和 Z 轴堆栈定位，请转到 焦点策略 选项卡以选择 使用由磁贴设置定义的焦点表面/Z 值。替代方法可以使用其他焦点策略，但建议使用此设置以获得最一致的结果。</li>
		<li>在 "切片 "选项卡下，单击 "验证 位置"，然后为每个位置手动设置中央 Z 平面。" 选项" 子选项卡中的设置将按图像采集顺序，因此请在开始成像之前检查此项。要按选择位置的顺序获取图像，请取消选中" 平铺区域/位置 "和 "载体孔/容器 "复选框。选择将 场景拆分为单独的文件 以简化图像处理。</li>
		<li>确保将 "Z-Stack "选项卡设置为" 居中"，将范围输入到用户的首选项中，并选择 "最佳 "按钮以设置切片间隔。</li>
		<li>优化采集 模式、通道、聚焦策略、Z 堆栈和 图块 选项卡后，开始实验。</li>
	</ol></li>
	<li>图像处理<ol><li>使用批量图像处理方法，使用 扩展焦深 方法创建每个 Z 堆栈的最大投影。</li>
		<li>使用批处理图像处理方法，将最大投影文件导出为 16 位 TIFF 图像。将压缩设置为 "无 "，并确保选中 "原始数据 "。所得图像应仅为表达脂质标志物的荧光通道的最大投影灰度TIFF。</li>
	</ol></li>
</ol>7. 细分和量化
注意:LipidUNet程序在96孔板的40倍图像上进行训练。强烈建议使用使用40倍物镜获得的图像。
<ol><li>安装 LipidUNet 软件。LipidUNet可以从以下GitHub存储库下载:https://github.com/RPEGoogleMap/LipidUNet</li>
	<li>识别代表尼罗河红、Bodipy 或 APOE 的 TIFF 图像，并根据所使用的方法将它们移动到名为 Nile_Red、Bodipy 或 APOE 的目录中名为 imgs 的文件夹中。 注意:LipidUNet 程序必须使用确切的命名约定才能识别目录。</li>
	<li>打开 LipidUNet 软件（图 4）。</li>
	<li>在软件的 "预测 "选项卡中，通过单击省略号并导航到命名目录来选择相关目录（Nile_Red、Bodipy 或 APOE）。通过检查类条目来确认 LipidUNet 程序已正确识别图像。</li>
	<li>为算法选择介于 0.01 和 0.99 之间的概率阈值。较高的值将消除更多的误报，但可能会导致更多的漏报，较低的值可能会引入更多的误报，同时消除更多的漏报。默认值为 0.65，建议使用。</li>
	<li>单击 预测。 注意:软件将自动循环访问所有映像，并在所选目录中创建一个名为predicted_masks的新文件夹。</li>
	<li>使用掩模分析工具循环访问生成的掩模，并提供来自 掩模 图像的阈值脂质沉积的定量计数。</li>
	<li>分析生成的计数数据以比较治疗条件。</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65503/65503fig04.jpg"> 图 4:LipidUNet 用户界面。 LipidUNet软件为训练数据目录选择不同的部分，其中脂质沉积物的图像已被正确识别;模型权重目录，该目录由训练数据生成;以及用户将在其中输入其图像以进行分割的预测数据目录。 <a href="https://www.jove.com/files/ftp_upload/65503/65503fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

建模和分析视网膜变性

<ol>
	<li>Kanow, M. 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=Biochemical+adaptations+of+the+retina+and+retinal+pigment+epithelium+support+a+metabolic+ecosystem+in+the+vertebrate+eye.">Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye.</a> eLife. 6, e28899 (2017).</li><li>Adijanto, 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=The+retinal+pigment+epithelium+utilizes+fatty+acids+for+ketogenesis.">The retinal pigment epithelium utilizes fatty acids for ketogenesis.</a> The Journal of Biological Chemistry. 289 (30), 20570-20582 (2014).</li><li>Farnoodian, 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=Cell-autonomous+lipid-handling+defects+in+Stargardt+iPSC-derived+retinal+pigment+epithelium+cells.">Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.</a> Stem Cell Reports. 17 (11), 2438-2450 (2022).</li><li>Curcio, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+drusen+in+age-related+macular+degeneration:+Biology+and+targeting+via+the+oil+spill+strategies.">Soft drusen in age-related macular degeneration: Biology and targeting via the oil spill strategies.</a> Investigative Ophthalmology &amp; Visual Science. 59 (4), AMD160-AMD181 (2018).</li><li>Cankova, Z., Huang, J. D., Kruth, H. S., Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passage+of+low-density+lipoproteins+through+Bruch's+membrane+and+choroid.">Passage of low-density lipoproteins through Bruch's membrane and choroid.</a> Experimental Eye Research. 93 (6), 947-955 (2011).</li><li>Curcio, C. 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=Esterified+and+unesterified+cholesterol+in+drusen+and+basal+deposits+of+eyes+with+age-related+maculopathy.">Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy.</a> Experimental Eye Research. 81 (6), 731-741 (2005).</li><li>Miyagishima, 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=AMPK+modulation+ameliorates+dominant+disease+phenotypes+of+CTRP5+variant+in+retinal+degeneration.">AMPK modulation ameliorates dominant disease phenotypes of CTRP5 variant in retinal degeneration.</a> Communications Biology. 4 (1), 1360 (2021).</li><li>Sharma, 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=Epithelial+phenotype+restoring+drugs+suppress+macular+degeneration+phenotypes+in+an+iPSC+model.">Epithelial phenotype restoring drugs suppress macular degeneration phenotypes in an iPSC model.</a> Nature Communications. 12 (1), 7293 (2021).</li><li>Farnoodian, 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=Cell-autonomous+lipid-handling+defects+in+Stargardt+iPSC-derived+retinal+pigment+epithelium+cells.">Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.</a> Stem Cell Reports. 17 (11), 2438-2450 (2022).</li><li>Hallam, D., 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+induced+pluripotent+stem+cell+patient+specific+model+of+complement+factor+H+(Y402H)+polymorphism+displays+characteristic+features+of+age-related+macular+degeneration+and+indicates+a+beneficial+role+for+UV+light+exposure.">An induced pluripotent stem cell patient specific model of complement factor H (Y402H) polymorphism displays characteristic features of age-related macular degeneration and indicates a beneficial role for UV light exposure.</a> Stem Cells (Dayton, Ohio). 35 (11), 2305-2320 (2017).</li><li>Sharma, R., Bose, D., Montford, J., Ortolan, D., Bharti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triphasic+developmentally+guided+protocol+to+generate+retinal+pigment+epithelium+from+induced+pluripotent+stem+cells.">Triphasic developmentally guided protocol to generate retinal pigment epithelium from induced pluripotent stem cells.</a> STAR Protocols. 3 (3), 101582 (2022).</li><li>Issa, P. C., Barnard, A. R., Herrmann, P., Washington, I., MacLaren, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+the+Stargardt+phenotype+in+Abca4+knockout+mice+through+inhibition+of+vitamin+A+dimerization.">Rescue of the Stargardt phenotype in Abca4 knockout mice through inhibition of vitamin A dimerization.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (27), 8415-8420 (2015).</li><li>GBD 2019 Blindness and Vision Impairment Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blindness+and+Vision+Impairment+Collaborators.+Causes+of+blindness+and+vision+impairment+in+2020+and+trends+over+30+years,+and+prevalence+of+avoidable+blindness+in+relation+to+VISION+2020:+the+Right+to+Sight:+an+analysis+for+the+Global+Burden+of+Disease+Study.">Blindness and Vision Impairment Collaborators. Causes of blindness and vision impairment in 2020 and trends over 30 years, and prevalence of avoidable blindness in relation to VISION 2020: the Right to Sight: an analysis for the Global Burden of Disease Study.</a> Lancet Global Health. 9 (2), e144-e160 (2021).</li></ol>

没有披露。

该协议提供了一种有效标记，成像和量化退行性眼病单基因和多基因 体外 疾病模型中脂质沉积的方法。基于人工智能的软件LipidUNet可应用于三种常见的脂质标志物，即APOE、Nile Red和BODIPY，并提供一种快速、自动的分析方法，使定量成为标准和无偏。
LipidUNet的主要局限性在于AI的训练数据集仅限于在96孔板中培养的细胞的40倍放大图像。作为训练图像集的结果，当前形式的LipidUNet仅限于分析40倍放大图像。该软件可用于分析除96孔板外在其他培养表面上培养的细胞的40倍图像，但应注意检查生成的输出掩模，以验证软件的准确阈值。需要更多的图像集（在不同的放大倍率下）来扩大它可以分析的样品/图像的范围。
该协议有几个关键步骤。在脂质标记步骤中，用户应确认他们选择的标记化合物（BODIPY，APOE，Nile Red）已有效标记其样品。成熟的RPE细胞通常色素沉着，这会损害抗体免疫染色的荧光信号。当荧光信号较弱或背景染色过多时，LipidUNet无法准确识别脂滴。出于类似的原因，必须使用正确选择协议自动成像步骤的采集设置。如果采集的图像质量较差，LipidUNet将难以正确屏蔽图像，因此定量将不准确（图6A-L）。最后，图像的后处理是一个重要的步骤，因为LipidUNet对软件的工作有特定的要求。
与使用手动阈值的脂质分析工作流程或斐济等软件中涉及自动阈值分析的技术相比，LipidUNet 在具有可变脂质沉积的图像中提供了无偏且可靠的分割，这反映在脂质颗粒鉴定中的小错误率（图 7）。该软件允许用户输入额外的训练图像，允许分析超出使用40倍放大镜的图像集，甚至是使用不同脂质标志物的图像集，如协议中所述。将来，该软件将被训练以分析3D图像，以便可以量化脂质沉积体积。将脂质沉积视为病理学主要因素的退行性眼病很普遍，随着老年人口的增加，预计病例还会增加13。正如我们在本协议中概述的那样，准确的疾病模型和有效的分析工具将允许开发新的治疗干预措施。

视网膜色素上皮（RPE）是位于眼睛后部与视网膜感光器相邻的单层细胞。RPE通过为光感受器提供代谢和结构支持，在维持正常视力方面起着至关重要的作用。健康的RPE细胞具有独特的六边形形态。它们通过紧密的连接连接，这使得RPE能够充当位于其基底侧的绒毛细血管和位于顶端的光感受器之间的屏障。为了维持视网膜生态系统，RPE 将关键代谢物（例如葡萄糖）运送到光感受器，以最大限度地减少 RPE 中的葡萄糖消耗1。由于这种限制，RPE依赖于其他代谢物来维持其代谢需求，包括脂肪酸，RPE通过β氧化将其转化为酮2。鉴于 RPE 倾向于利用脂肪酸（可能从感光器外段 （POS） 消化中回收）作为能量来源，RPE 中脂质加工途径的有害变化通常会导致或与单基因和多基因退行性视网膜疾病有关3。
年龄相关性黄斑变性（AMD）是一种导致RPE变性的多基因退行性眼病，也与RPE单层中的异常自噬和脂质代谢有关。功能失调的RPE单层无法处理POS和执行其他关键功能，导致细胞外（亚RPE）沉积，称为基础线性沉积物（BLinD），位于RPE和Bruch膜之间 - 这是AMD病理学的标志。BLinD的主要成分包括脂蛋白，其中最丰富的是载脂蛋白E（APOE）4。BLinD薄层的积累会导致软玻璃膜疣，这被认为是AMD5，6的临床症状。
一些小组已经表明，导致RPE功能障碍的干细胞衍生体外疾病模型具有亚RPE脂质积累7，8，9。Hallam等人（2017）由于CFH基因的多态性，从AMD高风险患者中产生了诱导多能干细胞衍生的RPE（iRPE）。iRPE显示玻璃膜疣积累，如APOE为标志，高风险RPE积累的沉积物大于低风险患者产生的iRPE10。
为了创建一个体 外 模型来概括AMD的细胞特征，如脂滴和玻璃膜疣沉积，使用先前发表的发育指导方案11建立了从患者血液样本生成的iRPE系。iRPE接受补体感受能力人血清（CC-HS），这是一种含有过敏毒素的溶液，模仿AMD的一个可能原因:增加替代补体信号传导8。使用常用的脂质和脂蛋白标记物APOE，尼罗河红和BODIPY测量产生的脂质沉积物的细胞和亚细胞沉积。通过这些标记物，显示通过CC-HS激活的补体信号传导加剧了iRPE细胞中的脂质积累8。
为了开发单基因视网膜退行性疾病的疾病模型，从Stargardt病患者开发了iRPE系，Stargardt病是一种由RPE中 ABCA4 基因突变引起的疾病。先前已经表明，当 ABCA4 被敲除时，A2E脂褐素（一种已知含有高水平磷脂和光依赖性脂质过氧化产物的细胞内沉积物）积聚在RPE12内。 ABCA4 敲除线与患者线一起开发，并且两者都经过 POS 喂养。Stargardt iRPE显示出POS吞噬作用诱导的病理学，表现出通过BODIPY染色定量的脂质积累增加。来源于 ABCA4 KO iPSCs的RPE进行CC-HS处理;BODIPY信号的定量显示Stargardt病模型中脂质处理存在缺陷9。
鉴于这些疾病的流行和对有效疗法的需求，以及上述相关疾病模型，有必要建立强有力的方法来量化潜在治疗的疗效。为了以客观、自动化和标准化的方式量化脂质沉积，我们创建了一个基于机器学习的软件 LipidUNet，以便与掩模分析工具配合使用时，可以使用常见的标记尼罗河红、BODIPY 和 APOE 快速有效地识别脂质沉积。然后可以分析和以图形方式分析和显示使用该分析管道获得的汇总统计数据，从而可以轻松比较治疗条件。该协议的原理图如图 1所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65503/65503fig01.jpg"> 图 1:方案示意图:RPE 细胞在 96 孔板上生长，并用活性人血清或纯化的牛外段攻击，以在体外模拟不同类型的视网膜变性。将RPE细胞固定并染色，以观察尼罗河红，BODIPY和APOE的脂蛋白沉积物。共聚焦显微镜用于获取荧光标记的脂质颗粒的Z堆栈，随后将其处理成2D最大强度投影。训练机器学习算法来识别和正确分割脂蛋白颗粒。生成包含颗粒计数和各种形状指标的汇总表，可用于后续绘图和统计分析。<a href="https://www.jove.com/files/ftp_upload/65503/65503fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Steriflip filter system</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;,5-Triiodo-L-thyronine</td>
			<td>Sigma</td>
			<td>T5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Bovine, Fraction V</td>
			<td>MP Biomedical</td>
			<td>160069</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 rabbit anti-goat IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21431</td>
			<td>APOE secondary antibody</td>
		</tr>
		<tr>
			<td>APOE primary antibody</td>
			<td>Millipore Sigma</td>
			<td>AB947</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY 493/503</td>
			<td>Invitrogen</td>
			<td>D3922</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Complement competent human serum</td>
			<td>Millipore Sigma</td>
			<td>S1-LITER</td>
			<td></td>
		</tr>
		<tr>
			<td>CTS N2 Supplement</td>
			<td>Life Technologies</td>
			<td>A13707-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td>Slide mounting media</td>
		</tr>
		<tr>
			<td>Glass Cover Slips #1 1/2 22 mm x 22 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>72204-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Microscope Slide 25 mm x 75 mm- 1.2 mm Thick</td>
			<td>Electron Microscopy Sciences</td>
			<td>71870-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0396</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Alpha</td>
			<td>Life Technologies</td>
			<td>12571-063</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential Amino Acids</td>
			<td>Life Technologies</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>Nile Red</td>
			<td>Sigma</td>
			<td>72485-100MG</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% Solution, EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Strep</td>
			<td>Life Technologies</td>
			<td>15140-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline 10x</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Rod Outer Segments (OS)</td>
			<td>InVision Bioresources</td>
			<td>98740</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S1888</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green Master Mix</td>
			<td>Bio-Rad</td>
			<td>1725274</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20 Ultrapure</td>
			<td>Affymetrix</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Life Technologies</td>
			<td>A14701SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>R&#38;D Systems</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

lipidunet-machine learning-based method of characterization and quantification of lipid deposits using ipsc-derived retinal pigment epithelium

视网膜色素上皮（RPE）是位于眼睛后部的单层六角形细胞。它为光感受器和脉络膜毛细血管提供营养和支持，对感光器外段（POS）进行吞噬作用，并以极化方式分泌细胞因子以维持外视网膜的稳态。由突变、衰老和环境因素引起的功能失调的 RPE 会导致其他视网膜层退化并导致视力丧失。退化 RPE 的一个标志性表型特征是细胞内和亚细胞内富含脂质的沉积物。这些沉积物是不同视网膜退行性疾病的共同表型。为了在 体外重现单基因视网膜变性的脂质沉积表型，从患者的成纤维细胞中产生了诱导多能干细胞衍生的RPE（iRPE）。Stargardt和迟发性视网膜变性（L-ORD）患者产生的细胞系用POS喂养7天以复制RPE生理功能，这在这些疾病中引起了POS吞噬诱导的病理。为了生成年龄相关性黄斑变性（AMD）的模型，这是一种与替代补体激活相关的多基因疾病，iRPE受到替代补体过敏毒素的挑战。使用尼罗河红、硼-二吡咯甲烷（BODIPY）和载脂蛋白E（APOE）对细胞内和亚细胞脂质沉积物进行了表征。为了量化脂质沉积物的密度，开发了一种基于机器学习的软件LipidUNet。该软件在培养表面上对iRPE的最大强度投影图像进行了训练。将来，它将被训练来分析三维（3D）图像并量化脂滴的体积。LipidUNet软件将成为发现减少疾病模型中脂质积累的药物的宝贵资源。

该协议提供了一种工作流程来鉴定尼罗河红，BODIPY和APOE染色的脂质沉积物。开发的软件可以自动识别和量化脂质沉积，并在优化概述的方案时表现最佳。其中包括成功分化的RPE（图3A）和低分化的RPE（图3B）的例子，因为细胞模型的质量极大地影响了正确图像分割的质量。
协议中描述的三个标记中的两个，尼罗红和BODIPY，被鉴定为小圆点，在荧光图像中明显明亮（图5和图6）。来自协议的"阳性"图像将是这些不同沉积物的适当识别（图5A-D和图5E-H）。由于染色弱（图6A-C和图6D-F）或由于高背景强度（图6G-I），"阴性"结果将显示不正确的图像分割，将背景荧光误认为沉积物。
APOE矿床具有各种大小和形状，看起来更椭圆形或不规则，而不是尼罗河红和BODIPY的圆形矿床。这些沉积物的点状性也较少，并且由于样品透化性的变化，沉积物之间的信号强度可能会有所不同。正确的识别将识别每个沉积物，包括那些饱和度较低的沉积物（图5I-L），而不正确的分割不会发现这些沉积物（图6J-L）。因此，优化染色和成像方法以避免剧烈变化非常重要。一种方法是在免疫染色时仔细注意样品透化步骤。为了优化荧光信号，可以在APOE固定和免疫染色之前裂解细胞，从而使APOE沉积物均匀饱和和更好地分割。
还提供了在96孔板以外的培养平台上成熟的细胞的分段图像。LipidUNet软件在跨孔培养的细胞图像上运行，虽然脂质沉积物被阈值化，但跨孔膜中的孔也被阈值化（图6M-O）。由于形状和大小的相似性，当前形式的LipidUNet软件将不分青红皂白地掩盖脂质沉积物和跨孔隙。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65503/65503fig05.jpg"> 图5:代表性结果。 （A，E，I） 96孔电镀RPE用Hoechst核染色（蓝色）和尼罗河红（品红色），BODIPY（绿色）或APOE（橙色）染色，并且是Z堆栈的最大强度投影。（乙、女、日）图像处理后LipidUNet软件的灰度输入图像。（中，由LipidUNet生成的掩码，其中所有沉积物都被正确识别。（深，高，中）每个掩蔽粒子的轮廓都有编号。这些标签允许将图像中的每个粒子连接到带有原始数据的传播中的条目。（A-D）显示尼罗河红染色，尽管信号较弱，但软件仍能够准确识别背景中的沉积物。（E-H）在BODIPY信号和背景之间显示出强烈的对比度，这是理想的。LipidUNet可以正确识别图像中的每个沉积物。（I-L）显示强烈的APOE信号，并表示该染色通常看到的信号饱和度的变化。尽管如此，图像分割能够识别每个APOE矿床的边界。<a href="https://www.jove.com/files/ftp_upload/65503/65503fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65503/65503fig06.jpg"> 图 6:次优结果。 （A，D，G，J，M） 96 孔镀 RPE 用 Hoechst 核染色（蓝色）和尼罗红（品红色）、BODIPY（绿色）或 APOE（橙色）染色，是 Z 堆栈的最大强度投影。（B，E，H，K，N）图像处理后LipidUNet软件的灰度输入图像。（C，F，I，L，O）由 LipidUNet 生成的不正确的掩码。红色圆圈表示软件错误地识别了脂质沉积物的位置。（A-C） 尼罗河红处理不正确，因为软件已将背景染色识别为沉积物。当图像中背景较高但脂质沉积很少时，这种情况可能会更频繁地发生。显示了两个BODIPY染色的例子:由于（D-F）弱的BODIPY染色引起的图像质量差和（G - I）具有高背景的强BODIPY信号。在这两种情况下，软件都无法将小的圆形脂质沉积物与围绕细胞核的背景圆形环区分开来。虽然应优化染色和成像以避免这些错误，但最新版本的 LipidUNet 针对这些图像进行了很大改进。（J-L）不正确的 APOE 分段。由于沉积物的大小和信号饱和度变化更大，因此该软件难以识别某些沉积物。（周一至奥）将RPE播种到Transwell上并用尼罗河红染色。这里显示了Z堆栈的一部分，其中包含尼罗河红脂质沉积物和跨孔孔。该软件无法区分两者，如包含跨孔的红色圆圈和指向尼罗河红矿床的绿色箭头所示。<a href="https://www.jove.com/files/ftp_upload/65503/65503fig06large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65503/65503fig07.jpg"> 图 7:遮罩工具比较 。 （A，B，C）具有可变脂质沉积量的96孔电镀RPE用尼罗红（红色）鉴定。使用三种不同的常见遮罩方法（查找最大值、最大熵和仁义熵）对图像进行遮罩，并与 LipidUNet 生成的掩码进行比较。原始图像伴随着脂质沉积物的手动计数，而掩模显示每种分割方法的预测计数。使用以下公式计算每种分割方法的平均错误率:mean[（|预测计数 - 手动计数|/手动计数） x 100]。与其他掩蔽方法相比，LipidUNet生成的掩模更准确地识别具有可变沉积的图像中的脂质沉积物（平均错误率:23%LipidUnet，1164%找到最大值，851%最大熵，203%仁义熵）。 <a href="https://www.jove.com/files/ftp_upload/65503/65503fig07large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>元件</td>
			<td>猫号</td>
			<td>股票集合</td>
			<td>最终会议</td>
			<td>毫升</td>
		</tr><tr><td>MEM 阿尔法</td>
			<td>12571-063</td>
			<td>那</td>
			<td></td>
			<td>500</td>
		</tr><tr><td>N2补充剂</td>
			<td>17502-048</td>
			<td>那</td>
			<td>1%</td>
			<td>5</td>
		</tr><tr><td>热灭活FBS</td>
			<td>SH30071.03</td>
			<td>那</td>
			<td>5%</td>
			<td>25</td>
		</tr><tr><td>NMEM NEAA</td>
			<td>11140-050</td>
			<td>10毫米</td>
			<td>0.01毫米</td>
			<td>5</td>
		</tr><tr><td>丙酮酸钠</td>
			<td>11360-070</td>
			<td>100毫米</td>
			<td>1毫米</td>
			<td>5</td>
		</tr><tr><td>青霉素-链霉素</td>
			<td>15140-122</td>
			<td>10000u/毫升</td>
			<td>100铌/毫升</td>
			<td>5</td>
		</tr><tr><td>牛磺酸</td>
			<td>T4571</td>
			<td>50毫克/毫升</td>
			<td>250微克/毫升</td>
			<td>2.5</td>
		</tr><tr><td>氢化可的松</td>
			<td>H6909</td>
			<td>18.1毫克/升</td>
			<td>20微克/升</td>
			<td>0.553</td>
		</tr><tr><td>T3</td>
			<td>T5516</td>
			<td>20微克/升</td>
			<td>0.013微克/升</td>
			<td>0.33</td>
		</tr><tr><td colspan="4">总体积，毫升</td>
			<td>548.383</td>
		</tr></tbody></table>表1:RPE-MM试剂组成。 RPE-MM的试剂和最佳浓度列表。

Watch this Scientific Journal Video about Unraveling the Pathogenesis of Age-Related Macular Degeneration and Discovering Potential Therapies at JoVE.com

作者聚焦:揭开年龄相关性黄斑变性的发病机制并发现潜在疗法 

影响眼睛视网膜色素上皮层的退行性眼病具有单基因和多基因起源。已经开发了几种疾病模型和软件应用程序LipidUNet来研究疾病的机制以及潜在的治疗干预措施。

基于脂质UNet-机器学习的脂质沉积物使用iPSC衍生的视网膜色素上皮表征和定量脂质沉积物的方法

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to ...

在这项研究中，我们旨在了解与年龄相关的宏观变性发病机制并找到潜在的治疗方法。这种用于脂滴分析的半自动管道为研究代谢和衰老疾病提供了一个强大的系统。我们的实验室专注于寻找治疗AMD的药物靶点，无论是使用旨在减少脂质沉积的遗传或小分子筛选。

lipidunet-machine-learning-based-method-characterization

Research

JoVE Journal

Biology

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

2.4K 次观看.  National Institutes of Health (NIH). 在这项研究中，我们旨在了解与年龄相关的宏观变性发病机制并找到潜在的治疗方法。这种用于脂滴分析的半自动管道为研究代谢和衰老疾病提供了一个强大的系统。我们的实验室专注于寻找治疗AMD的药物靶点，无论是使用旨在减少脂质沉积的遗传或小分子筛选。

视频: 作者聚焦:揭开年龄相关性黄斑变性的发病机制并发现潜在疗法 

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

科研

生物学

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

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

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

小鼠视网膜色素上皮细胞的原代培养

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

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

猪视网膜色素上皮细胞原代培养

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

小鼠原代视网膜色素上皮细胞的分离

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

使用高分辨率呼吸测定法实时分析原代人视网膜色素上皮细胞中的生物能量学

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

量化视网膜色素上皮吞噬作用和脂褐素积累

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

作者聚焦：改进的脂褐素模型和高度极化人类视网膜色素上皮培养物中外段吞噬作用能力的定量  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

改进脂褐素模型和高极化人视网膜色素上皮培养物中外段吞噬能力的定量

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...