这项研究工作得到了欧盟的资助。H2020-MSCA-765441），德国研究理事会（DFG;PA1751/8-1，10-1）和中国奖学金委员会。

符合德国动物保护法第4条的动物议定书由图宾根大学动物保护委员会（埃因里希通·费尔·蒂尔舒茨、蒂雷兹特利琴·迪恩斯特和拉蒂尔昆德）审查和批准:注册号AK02/19M）。在这项研究中，视网膜是从野生型（WT）和rd1小鼠中获得的，后者是遗传性视网膜变性15的一个特征良好的模型。老鼠被安置在标准的白色循环照明下，可以自由获得食物和水，并且无论性别如何使用。
1. 清单
<ol><li>为确保无菌条件，避免污染，使用 70% 乙醇清洁和消毒层气流罩。一定要让乙醇完全蒸发，防止视网膜培养物的陶醉。</li>
	<li>使用前自动切割工具（如剪刀、钳子和眼科显微镜刮勺）。</li>
	<li>在无菌条件下，提前在层流罩下准备以下介质:Basal R16 介质 （BM） （可存储在 4 °C 4 周）、BM 与 20% 胎儿小腿血清 （FCS） （同一天使用），BM 与 0 .12% 蛋白酶 K （44 mAnson U/mg） 溶液 （当天使用） 和完整的 R16 介质与补充剂 （CM） 如 Romijn16所描述 （可存储在 4 °C 3 周） （见表 S1， S2，和S3）。</li>
	<li>在 37 °C 预热蛋白酶 K 溶液以激活它，并在第 2.5 步中使用它。</li>
</ol>2. 准备
<ol><li>在产后日（P）5通过斩首牺牲 rd1/WT动物。对于超过P11的动物，根据当地动物保护条例，使用二氧化碳 和/或宫颈错位。</li>
	<li>根据动物的年龄，在诱导之前，如果需要，使用钳子打开眼睑，非常小心地分离眼睑，而无需触摸或划伤下面的眼睛。</li>
	<li>使用弯曲的钳子在立体镜下快速诱导眼睛。</li>
	<li>在室温下将眼睛在 BM 中孵育 5 分钟 （RT）。</li>
	<li>在预热的BM中孵化眼睛，在37°C下用0.12%的蛋白酶K孵育15分钟。</li>
	<li>在层压气流罩内执行以下步骤，以确保无菌条件。要灭活蛋白酶 K，将眼睛转移到含有 20% FCS 的 BM，并在 RT 孵育 5 分钟。</li>
	<li>在 RT 的培养皿中，在立体镜下解剖眼睛，无菌地在 RT 中含有新鲜的 BM。时间越长，解剖视网膜就越难，眼睛变得很软。<ol>
<li>用钳子，从视神经保持眼睛。使用细剪刀，在角膜上做一个小切口，创建2个边缘，从角膜，胆汁和硬皮可以轻轻地去皮使用2对细钳（图1 步骤A–C）。或者，使用窄口径的管子将第一个切口切入角膜，然后将其中一把剪刀刀片插入开口。</li>
		<li>用细钳抓住镜头。将第二对钳子垂直于第一对，以便第一个钳子在第二个磁盘的 2 个刀柄之间。拉从眼杯中提取镜头。如果静脉和胆体仍然附着在视网膜上，小心地将其移除（图1 步骤D）。 注意:步骤 2.7.1 和 2.7.2 需要练习，并确保谨慎不要损坏网膜。</li>
		<li>将垂直于边缘的网状网状物切成四点，形成四叶三叶草形状（图1 步骤E–F）。</li>
		<li>使用 1 mL 尖端广泛切割底座的移液器，将网状膜放在悬挂的介质中，并将其转移到放置在 6 井培养板中的培养皿滤芯插入物中。RPE 层应面向膜（图 1 步骤 G）。</li>
		<li>使用移液器，小心地从插入物中去除多余的介质。</li>
		<li>从井的两侧，每口井添加1毫升CM，在37°C的无菌孵化器中孵化，二氧化碳2为5%。不要将小膜浸入介质中，因为这会减少氧合并导致组织退化。去种植物应保持在液体和空气之间的界面，只覆盖由水表面张力产生的薄薄的液体薄膜。</li>
	</ol>
</li>
	<li>在前 48 小时内保持视网膜外植不受干扰，以便于去除程序后恢复。</li>
	<li>每隔两天更换一次介质（48 小时）。从每口井中丢弃 700 μL 的介质，并在井中加入 900 μL 的新鲜 CM。这样，蒸发失去的介质量被恢复，视网膜外植保留了前48小时产生的一些神经保护因子。</li>
	<li>将去除的介质与培养物一起在单独和封闭的微中流管中孵化，以控制和评估可能的污染（即介质颜色的变化）。 注:视网膜外植可以在培养中保存至少2周12。</li>
</ol>3. 栽培后
注:外植可用于不同的实验应用（西斑、组织学、全坐骑、基因分析、电生理学）。根据应用，有机视网膜外植物可以捕捉冷冻、解冻或准备冷冻。下面的步骤描述了神学准备。
<ol><li>用4%的副甲醛（PFA）进行45分钟的固定，然后进行渐进的蔗糖低温保护（10分钟10%蔗糖，20分钟20%，室温（RT）或夜间（开）在4°C时2小时30%。将这些缓冲器直接添加到井中。</li>
	<li>切开视网膜外植物周围的膜。</li>
	<li>将膜和视网膜组织嵌入介质中，用于冷冻组织。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61868/61868fig01.jpg"> 图1:器官视网膜外植培养物的编制分步程序。（A） 小鼠眼睛被诱导并转移到蛋白酶K溶液中，以便将硬盘和胆汁从视网膜和RPE中分离出来。引入了囊泡/巧克力层的一个小切口。（B） 两个钳子用于剥去硬皮/胆汁层。（C） 在剥皮过程中可以看到黑色巧克力层。下划线的深色视网膜色素上皮 （RPE） 仍然附着在眼球上。硬骨和胆汁与视神经一起被切除。（D） 镜头和钳子用钳子提取。 剩余的胆小的身体被移除。（E） 网状网膜保持碗状形状。（F） 为了平整在盘子中培养的网状网膜，用剪刀在网状物周围等距离内进行4次切割，使其形成三叶草状的形状。使用切割的 1 mL 移液器尖端，将网膜培养物转移到插入 6 井板中的膜培养物中。网状网膜仍然保留一些碗形。然而，在去除视网膜周围的多余的液体后，它将展开到平面结构。（G） 在培养膜设置中，网状膜培养物位于多孔聚碳酸酯 （PC） 膜上，位于完整的 R16 介质溶液之上。为了确保生存能力，该培养必须保持在一个潮湿的无菌孵化器中，温度为37°C，二氧化碳为5%，并且每48小时更换<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/61868fig01large.jpg" style="font-size: 14px;" target="_blank">一</a>次介质。

作者没有什么可透露的。

所提交的协议描述了小鼠网状体的有机去除培养物，在定义的R16介质中具有完整的RPE，不含血清和抗生素。该协议最初开发于20世纪80年代末7，28，从那时起，它一直在不断完善6，11，12。值得注意的应用包括研究遗传性视网膜退化的机制和视网膜保护药物的鉴定23，29，30。
要想进行成功的实验，需要考虑一些重要的考虑因素。这里有一些重要的故障排除点，以帮助提高文化的质量。首先，视网膜文化可能表现出过度折叠和/或玫瑰花环形成31。这可以通过在去除过程中用钳子触摸直膜引起。此外，必须从除法植物中完全去除胆体，因为这样可以增加培养过程中的视网膜折叠。其次，在将小雷管转移到井板的悬垂滴中，如果网状网膜朝下面朝下，将其放在悬挂在移液器尖端的掉落处，并非常轻轻地将介质推入和推出尖端（无需分离悬垂滴）以翻转网状体。最后，如果 RPE 仍然附着在囊泡上，并且从网状网膜上分离，则很可能是由于硬盘消化不足造成的。在与来自老动物或非啮齿动物（如猪）的眼睛一起工作时，这个问题可能特别重要，并且可以通过增加蛋白酶K浓度来解决。
进行有机视网膜外植培养是一个复杂的过程，需要充分的培训和经验。缺乏培训可能导致视网膜外植质量的变异。出于这些原因，必须监测和验证可行性和可重复性，例如，用 TUNEL 检测来描述细胞死亡率。使用无抗生素介质使视网膜外植物容易受到细菌和真菌的污染。为了尽量减少这种风险，我们建议在真正无菌的条件下特别小心工作。 体外视网膜培养的另一个限制是与 体内 视网膜（例如，胆囊和视网膜供血、氧气和葡萄糖水平、眼内压力、体外组成）相比，物理化学环境的差异。一个连续的灌注系统，也许嵌入到一个专门的生物反应器32可以使这个模型更接近 体内 的条件。此外，视网膜解剖期间视神经的轴切除术会导致结节细胞死亡，从而诱发应激反应8。因此，建议在受特定操作或治疗之前，让去种植物 在体外 至少2天适应栽培条件。
上述方法通常在不成熟的视网膜组织上进行，在体外7、33中存活4周。然而，该程序是针对各种应用量身定制的，包括成人直膜的培养。虽然不同已发布的方法描述的成人视网膜的隔离没有其RPE34，35， 孵化与木瓜蛋白溶液长达1小时在37 °C之前解剖允许RPE保持连接到视网膜，即使来自成人小鼠36.
无血清介质和化学定义的体外环境为完全定义和可重复的实验条件提供了操作。因此，有机视网膜外植培养是眼科和神经科学领域的宝贵工具，曾用于研究视网膜疾病37、视网膜发育38、39、视网膜干细胞治疗40、基因改造41和药理筛选。作为药物测试的一个具体例子，在这里我们使用视网膜外植培养物来测试cGMP模拟（CN003），已知可以减少遗传视网膜疾病动物模型中的光感受细胞死亡23（图3B）。该技术的另一个可能应用在图3C中描述，它说明了如何利用组织环境的精确控制来模拟糖尿病状况24。由于在整个培养期组织结构的保存，有机视网膜外植培养也适合电生理学研究。使用补丁夹记录42和多电极阵列（MEA）记录33，43对视网膜外植物的神经元功能进行了研究。后者允许同时记录神经元人群的电活动，并利用它来描述培养条件下的光接受器和结节细胞功能。从更广的角度看，有机外植培养系统也可以应用于临床前研究，其中外植培养用于测试体温过低44的疗效。
有机体外植培养技术相对简单，与 体内 实验相比，成本更低，耗时更短，避免了与活体动物研究相关的伦理问题。对实验条件的精确控制以及RPE和组织复杂性的保存，使该方法成为提高我们对视网膜生理学和病理生理学知识的宝贵工具，并能够进行大量实验应用。

在眼科研究中，有多种模型可用于研究视网膜，包括原发性视网膜细胞培养、视网膜衍生细胞系、视网膜器官，以及体内动物模型1、2、3、4、5。然而，这些模型中的每一个都有缺点。例如，细胞在孤立中生长，而网膜是一个复杂的网络，具有多种细胞对细胞的相互作用。因此，与整个组织中观察到的行为相比，孤立细胞培养的行为可能是人为的。这个问题可以部分地用体外分化视网膜器官来补救，可用于研究发育和基础生物学6。然而，从今天起，视网膜器官的生成仍然是耗时的，劳动密集型的，并遭受可重复性问题，需要大量的进一步发展工作之前，器官可用于转化视网膜研究。最后，对活体动物的研究，虽然可以说是最接近眼科研究要求的模型，但与强烈的伦理问题有关。细胞培养系统的效率与体内动物模型的真实情况之间的一个很好的折衷方案是有机视网膜外植培养。这种文化也减少了动物的痛苦，因为没有进行体内干预。
已经描述了几种方法来培育来自不同物种5，7，8的视网膜外植。我们的协议描述了一种隔离小鼠神经神经质及其视网膜色素上皮（RPE）的技术。这项技术也将适合大鼠视网膜培养9。神经神经质培养及其 RPE 对于成功至关重要。RPE对视网膜具有基本功能:输送养分、离子、水、吸收光线和防止光氧化，将全跨视网膜重新异构成11-cis-视网膜，这对视循环、棚光感受膜的幻象形成以及视网膜结构完整性的基本因素分泌至关重要。维护 RPE 可成功开发光感受器的外部和内部部分，使光网膜在较长时间内保持活力。下文描述的程序至少保存了两个星期12的直肠组织和生理特征。此外，在无血清、无抗生素的介质中培养有机视网膜外植可避免存在未知物质，并能够直接解释结果12。
有机视网膜外植培养对于提高我们对视网膜发育和退化的知识至关重要。我们在这里表明，它们也是药理学筛查的有用工具，它们可以用来模拟各种视网膜疾病，包括糖尿病视网膜病变。

<table><tbody><tr><td>Biotin</td><td>Sigma</td><td>B4639</td><td></td></tr><tr><td>(+/-)-&amp;alpha;-LipoicAcid (=Thiotic acid)</td><td>Sigma</td><td>T1395</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>B4639</td><td></td></tr><tr><td>CDP-Choline-Na</td><td>Sigma</td><td>30290</td><td></td></tr><tr><td>Corticosterone</td><td>Sigma</td><td>C2505</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt; &amp;times; 5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>C8027</td><td></td></tr><tr><td>DL-Tocopherol</td><td>Sigma</td><td>T1539</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma</td><td>E0135</td><td></td></tr><tr><td>FCS</td><td>Sigma</td><td>F7524</td><td></td></tr><tr><td>Filtropur BT100, 1L, 0.2&amp;micro;m</td><td>SARSTEDT</td><td>83.3942.101</td><td>for Basal Medium</td></tr><tr><td>Forceps&amp;nbsp;</td><td>F.S.T</td><td>15003-08</td><td></td></tr><tr><td>Glutamine</td><td>Sigma</td><td>G8540</td><td></td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G6013</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma</td><td>I6634</td><td></td></tr><tr><td>L-CysteineHCl</td><td>Sigma</td><td>C7477</td><td></td></tr><tr><td>Linoleic Acid</td><td>Sigma</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma</td><td>L2376</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2 &lt;/sub&gt;x 4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M5005</td><td></td></tr><tr><td>Na-pyruvate</td><td>Sigma</td><td>P3662</td><td></td></tr><tr><td>NaSeO&lt;sub&gt;3 &lt;/sub&gt;x 5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S5261</td><td></td></tr><tr><td>Ophthalmic microscope scaping spoon</td><td>F.S.T.</td><td>10360-13</td><td></td></tr><tr><td>Progesteron</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Proteinase K&amp;nbsp;</td><td>MP Biomedicals</td><td>21935025</td><td>44 mAnson U/mg</td></tr><tr><td>R16</td><td>Gibco</td><td>07491252A</td><td></td></tr><tr><td>Retinol</td><td>Sigma</td><td>R7632</td><td></td></tr><tr><td>Retinyl acetate&amp;nbsp;</td><td>Sigma</td><td>R7882</td><td></td></tr><tr><td>Scissors</td><td>F.S.T</td><td>15004-08</td><td></td></tr><tr><td>Sterile filter 0.22&amp;micro;m</td><td>MILLEX GP</td><td>SLGP033RS</td><td>for supplements</td></tr><tr><td>T3&amp;nbsp;</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>Tocopherylacetate</td><td>Sigma</td><td>T1157</td><td></td></tr><tr><td>Transferrin</td><td>Sigma</td><td>T1283</td><td></td></tr><tr><td>Transwell permeable supports</td><td>Corning</td><td>3412</td><td></td></tr><tr><td>Vitamin B1</td><td>Sigma</td><td>T1270</td><td></td></tr><tr><td>Vitamin B12</td><td>Sigma</td><td>V6629</td><td></td></tr><tr><td>Vitamin C</td><td>Sigma</td><td>A4034</td><td></td></tr></tbody></table>

long-term, serum-free cultivation of organotypic mouse retina explants with intact retinal pigment epithelium

在眼科研究中，神经神经内膜的 体外 模型非常需要。在这里，我们提出了一个详细的协议，为细胞图培养小鼠 神经视网膜与完整的视网膜色素上皮（RPE）。根据研究问题，视网膜可以从野生类型的动物或疾病模型中分离出来，例如研究糖尿病视网膜病变或遗传性视网膜退化。产后早期2-9动物的眼睛在无菌条件下被诱导。它们部分被消化在蛋白酶K中，以便将胆汁从RPE中分离。在立体镜下，角膜形成一个小切口，形成两个边缘，从其中胆汁和硬骨可以轻轻地从 RPE 和神经精子剥离。然后，将镜头拆下，眼杯切成四点，使其具有四楔形，类似于三叶草叶。组织最终被转移到一个挂滴到细胞培养插入持有聚碳酸酯培养膜。然后，这些培养物在R16介质中保持，没有血清或抗生素，在完全定义的条件下，每隔一天发生一次中等变化。
所述程序使小膜能够隔离，并维持其正常的生理和组织背景，至少持续2周的培养期。这些功能使有机视网膜外植培养成为具有高预测价值的优秀模型，用于研究视网膜发育、疾病机制和电生理学，同时实现药理学筛查。

遵循协议后，解剖和培养的视网膜除外植物保留其正常的组织结构，具有不同的层，从RPE到结节细胞层（GCL），如图2所示。外层核层（ONL）和内部核层（INL）的大小在2-3周内基本保持稳定，如果培养期延长到4周及以后，细胞流失缓慢，这些层逐渐变薄。相比之下，在GCL中，由于视神经的轴切除术，通常在培养的头4天内观察到明显的变薄。之后，GCL中剩余的细胞群（大部分为置换的阿马格林细胞）将继续存活3-4周17、18、19。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61868/61868fig02.jpg"> 图2:视网膜外植培养中发现的细胞类型。P11的视网膜外植培养来自rd1突变体（A）和WT动物（B），显示核染色与DAPI（左，蓝色），棒光感受器（中，红色）和穆勒细胞（右，绿色）。核染色突出了视网膜的所有主要细胞层，如视网膜色素上皮（RPE）、外核层（ONL）、内核层（INL）和结膜细胞层（GCL）。核层中的特定细胞类型，如棒状细胞和穆勒细胞，分别用α逮捕素26和谷氨酰胺合成酶27抗体免疫。（C） 显示整个   rd1小鼠视网膜的全长部分，DAPI 染色突出了视网膜的一致性、集成和发展性。这些视网膜被培养了6天。程序描述:从rd1或WT动物中提取的雷蒂娜和RPE在P5被隔离，并按照协议所述进行培养，直到P11每48小时发生一次中等变化。文化在P11时用4%的PFA固定，并进行了冷冻剖析。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/61868fig02large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本</a>
无血清介质和持续的 体外 环境使能够完全控制实验条件。在这里，我们为本协议的具体应用提供两个示例。 第一个例子说明了使用视网膜外植进行药物测试或筛选的可能性。有机视网膜外植培养物是从野生类型 （WT） 和rd1小鼠模型中准备的。后者是视网膜变性15的一个特点良好的模型。在rd1小鼠网膜中，ONL 退化是由棒光感受器6、20中异常高的 cGMP 水平触发的。过量的cGMP导致循环核苷酸门离子通道（CNGCs）和cGMP依赖蛋白激酶（PKG）的活性增加，导致细胞死亡21。用结构类似于cGMP（环核苷酸#3的31小鼠视网膜的处理:针对 PKG 和 CNGC 的 CN003 进行了测试。在 P5 去除后，遵循图 3A中描述的治疗模式。在P11中，外植培养物固定在4%PFA，并准备进行冷冻切除（图3A）。为了评估从治疗，非治疗（NT）和WT标本组织学部分的细胞死亡，进行了终端脱氧核苷酸转移酶dUTP尼克结束标签（TUNEL）检测22。TUNEL标记细胞的分析显示，在未经处理的标本的UNL中，死亡细胞的比例很高，而CN003在浓度为50μM23（图3B）时保护了第1个小鼠光感受器。
糖尿病的常见并发症是糖尿病视网膜病变，一种致盲疾病，很难在动物模型5中忠实地繁殖。第二个例子突出了使用有机视网膜外植培养物来描述视网膜细胞在模拟2型糖尿病（T2DM）24的条件下的生存能力。在这里，我们使用20mM的糖化抑制剂2-脱氧葡萄糖（2-DG）25，并管理它到培养介质24小时从P10到P11。我们表明，将WT视网膜外植受制于这种体外模拟糖尿病疾病会导致视网膜（图3C）广泛的神经元细胞死亡。然后，这种范式可以反过来用于研究退行机制或在糖尿病背景下测试视网膜保护治疗。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61868/61868fig03.jpg"> 图3:两个应用有机视网膜外植培养物的例子。 （A） 程序描述: 来自rd1或WT动物的视网膜和RPE在产后日（P）5被隔离，并如上所述进行培养，每48小时发生一次中等变化。在 P7 和 P9 中，废中等被丢弃，在板中加入浓度为 50μM 的含有活性化合物的新鲜 CM。文化在P11时用4%的PFA固定，并进行了冷冻剖析。（B） 对rd1小网膜进行复合测试。部分来自WT，治疗（003）和非处理（NT）有机视网膜外植培养物。图恩尔检测（红色）被用作细胞死亡的标记。核染色与DAPI（蓝色）。条图中显示的定量说明了WT、rd1 NT 和rd1 003 条件下死亡细胞的百分比。用化合物003治疗显著减少rd1光感受器细胞死亡。（C） 使用 2 脱氧葡萄糖 （2-DG） 治疗方式模拟 WT 视网膜上的 2 型糖尿病 （T2DM）。在NT和T2DM标本上进行了TUNTL检测。量化表明细胞死亡显著增加。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/61868fig03large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本</a>
表S1。  <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/Table%20S1%20-%20Basal%20Medium.doc" target="_blank">请单击此处下载表</a>
表 S2。  <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/Table%20S2%20-%20Supplements.doc" target="_blank">请单击此处下载表</a>
表 S3。  <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61868/Table%20S3%20-%20Complete%20Medium.doc" target="_blank">请单击此处下载表</a>

Watch this Scientific Journal Video about Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium at JoVE.com

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

该协议描述了小鼠 神经视网膜的细胞图外植，与视网膜色素上皮（RPE）一起在R16定义的介质中培养，不含血清和抗生素。与 体内 实验相比，这种方法性能相对简单、成本更低、耗时，可适应多种实验应用。

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

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

long-term-serum-free-cultivation-organotypic-mouse-retina-explants

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.5K 次观看.  University of Tübingen. 该协议描述了小鼠 神经视网膜的细胞图外植，与视网膜色素上皮（RPE）一起在R16定义的介质中培养，不含血清和抗生素。与 体内 实验相比，这种方法性能相对简单、成本更低、耗时，可适应多种实验应用。

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

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

器官型培养的全厚成年猪视网膜

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

科研

神经科学

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

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 age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

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

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

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 ...

发育生物学

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

在体内的器官型小鼠视网膜Wholemount文化

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

生物学

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

<meta charset="utf8"></head><body>生成人类患者 iPSC 衍生的视网膜类器官以模拟色素性视网膜炎

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

生成人类患者 iPSC 衍生的视网膜类器官以模拟色素性视网膜炎

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

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

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

新型人类组织特征视网膜培养技术的特征

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

医学

Organotypic Retinal Explant Cultures from Macaque Monkey

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) pathway in photoreceptor cells causes photoreceptor cell death, especially in models harboring phosphodiesterase 6b (PDE6b) mutations. Previous studies on RD have used mainly murine models such as rd1 or rd10 mice. Given the genetic and physiological differences between mice and humans, it is important to understand to which extent the retinas of primates and rodents are comparable. Macaques share a high level of genetic similarity with humans. Therefore, wild-type macaques (aged 1-3 years) were selected for the in vitro culture of retinal explants that included the retina-retinal pigment epithelium (RPE)-choroid complex. These explants were treated with different concentrations of the PDE6 inhibitor zaprinast to induce the cGMP-PKG signaling pathway and simulate RD pathogenesis. cGMP accumulation and cell death in primate retinal explants were subsequently verified using immunofluorescence and the TUNEL assay. The primate retinal model established in this study may serve for relevant and effective studies into the mechanisms of cGMP-PKG-dependent RD, as well as for the development of future treatment approaches.

Retinal explants obtained from wild-type macaques were cultured in vitro. Retinal degeneration and the cGMP-PKG signaling pathway was induced using the PDE6 inhibitor zaprinast. cGMP accumulation in the explants at different zaprinast concentrations was verified using immunofluorescence.

猕猴的器官型视网膜外植体培养物

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

培养的原代鼠 RPE 的表征和形态动力学

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

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

作者聚焦：从成年小鼠中有效分离原代视网膜色素上皮细胞用于视网膜病变研究

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

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

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

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

摘要

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器官型培养的全厚成年猪视网膜

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定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

在体内的器官型小鼠视网膜Wholemount文化

生成人类患者 iPSC 衍生的视网膜类器官以模拟色素性视网膜炎

生成人类患者 iPSC 衍生的视网膜类器官以模拟色素性视网膜炎

新型人类组织特征视网膜培养技术的特征

猕猴的器官型视网膜外植体培养物

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

长期，无血清培养有机小鼠视网膜外植与完整的视网膜色素上皮

摘要

探索更多视频

器官型培养的全厚成年猪视网膜

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

在体内的器官型小鼠视网膜Wholemount文化

生成人类患者 iPSC 衍生的视网膜类器官以模拟色素性视网膜炎

生成人类患者 iPSC 衍生的视网膜类器官以模拟色素性视网膜炎

新型人类组织特征视网膜培养技术的特征

猕猴的器官型视网膜外植体培养物

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型