这项研究由 BrightFocus 基金会 (DS) 部分资助。

涉及动物问题的程序已获得匹兹堡大学机构动物保育和使用委员会 (IACUC) 的批准。
1. 准备解决方案
<ol><li>通过补充 Dulbecco 修饰的鹰培养基 (DMEM)、高葡萄糖、10% 胎牛血清 (血清)、1% 青霉素/链霉素、2.5 毫米 l-谷氨酰胺和1x 内存不必要的氨基酸, 制备生长培养基。预热的媒体在37摄氏度孵化器使用前。</li>
	<li>准备 2% (酶) II 工作解决方案:<ol>
<li>在300µL HEPES 缓冲盐水 (50 毫米 HEPES/KOH pH 7.4, 150 毫米氯化钠) 中溶解30毫克的酶 ii 酶, 制备100毫克/毫升的库存溶液。这个体积是 3-4 小鼠, 它可以放大或下降的基础上的小鼠数量的实验)。该股票可以储存至少1周在4摄氏度。</li>
		<li>2% 酶的工作解决方案, 加入300µL 100 毫克/毫升酶 II 库存到1.2 毫升的无菌 DMEM, 高葡萄糖和过滤溶液通过0.22 µm 过滤器。此步骤应在无菌条件下层流柜内进行。预热前准备酶 II 在37°c 孵化器使用前。</li>
	</ol>
</li>
</ol>2. 获取鼠标眼睛
<ol><li>使用任何已批准的方法弄死鼠标, 并将其放在吸水垫上。</li>
	<li>戴上手套, 把拇指和食指放在眼睛周围, 轻轻地推着皮肤 proptose 眼球。</li>
	<li>在皮肤和眼球之间插入尖角剪刀, 小心地剪掉眼睛。为了避免污染, 在70% 乙醇中简单地蘸上眼睛。</li>
	<li>立即把眼睛在磷酸盐缓冲盐水 (PBS) 的培养皿。</li>
	<li>重复步骤 2.2-2.4 以移除第二只眼睛。</li>
</ol>3. 眼部解剖
<ol><li>在解剖显微镜下小心地从眼睛中取出结缔组织。这可以做在 PBS 或干培养皿。<ol>
<li>使用缝合钳从眼球中提起结缔组织, 用 Vannas 剪刀将其切开。不要切开巩膜, 因为如果巩膜被切开, 几乎不可能获得完整的后眼罩。</li>
		<li>清洗结缔组织后, 将所产生的眼球放在 PBS 中进行下一个洗涤步骤。 注: 以下步骤应在无菌条件下层流柜内进行。</li>
	</ol>
</li>
	<li>两次洗眼睛前预热37°c DMEM (高葡萄糖)。用镊子小心地将眼球转到预热的37摄氏度 DMEM (高葡萄糖)。在新的盘子中吸入培养基并将眼球转移到新鲜的培养基上。</li>
	<li>吸 DMEM (高葡萄糖) 培养基和孵化的眼睛在预热 2% (重量/卷) 酶 II 工作解决方案45分钟在37°c 孵化器。 注意: 在以下所有步骤中, 在生长培养基中放置和操作眼睛或眼罩, 这将保护组织并为解剖提供良好的培养基。</li>
	<li>移除酶 II 溶液, 通过吸旧介质并加入新的预热37°c 生长介质 (图 1a), 在生长培养基中两次冲洗眼睛。 注: 酶后, 眼球变软。</li>
	<li>用钳夹住眼睛, 用 Vannas 剪刀在层流柜内的解剖显微镜下切开每只眼睛周围的青锯。<ol>
<li>保持眼球在溶液中, 小心地移除前角膜通过扩大切口周围的锯缘, 并拉前角膜离开后, 切口已经完成。</li>
		<li>用齿钳 (图 1b, 1c) 轻轻拉出眼罩, 取出晶状体囊和相关的虹膜色素上皮。</li>
	</ol>
</li>
	<li>孵化后眼罩在生长培养基中的20分钟, 在37摄氏度, 以促进分离的神经视网膜从视网膜色素。</li>
	<li>在层流柜内放置解剖显微镜下, 将后眼罩切成4瓣。切口应该足够长以使眼罩平坦, 但短到足以保持花瓣连接 (图 1d)。<ol>
<li>扁平化的眼罩和消除神经视网膜通过非常轻轻地拉它与钳, 而持有其余的 RPE-脉络膜-巩膜复合体与另一个钳 (图 1e)。从边缘而不是从中心开始拉。</li>
	</ol>
</li>
	<li>将所有的被扎营的组织转移到一个新的无菌培养皿中, 预热37摄氏度生长培养基。</li>
</ol>4. 原发性视网膜色素上皮细胞的分离
<ol><li>在解剖显微镜下, 用显微外科月牙刀在刷的基底膜 (膜) 上轻轻剥离出完整的视网膜色素片, 用超细钳夹住一瓣。放置在层流流柜中 (图 1f)。<ol>
<li>使用 P200 微将视网膜色素表转移到含有生长培养基的新型无菌培养皿中。当转移视网膜色素表时, 通过吹打多次湿润吸管提示和生长培养基, 以防止组织在尖端内黏附。视网膜色素从脉络膜可以明显区分: 视网膜色素看起来更褐色, 而脉络膜更暗, 看起来黏糊糊和蓬松。</li>
		<li>或者, 使用酶消化和温和离解没有钳, 以分离视网膜色素从脉络膜14。</li>
	</ol>
</li>
	<li>用预热的37°c 生长培养基在无菌培养皿中冲洗 2-3 倍的 RPE 片。每个洗涤步骤后, 仔细收集视网膜色素表, 同时避免其他组织, 如视网膜碎片或脉络膜。在最后洗涤的结尾收集视网膜色素表与 P200 微并且安置他们在一个新的1.5 毫升离心管。 注: 没有必要离心的视网膜色素表: 他们将在1分钟内的重力沉积。</li>
	<li>使用 P200 微移除额外的生长介质。并用重悬新鲜的预预热生长培养基中的细胞, 并用 P200 微轻轻 triturate 它们。避免气泡的形成。单细胞悬浮是理想的, 但也避免太多的研磨, 否则视网膜色素细胞无法生存。我们建议非常温和的研磨 40-50 倍。</li>
</ol>5. 培养视网膜色素上皮细胞
<ol><li>将细胞印在培养板上。<ol>
<li>如果视网膜色素上皮细胞的下游应用需要保留其极性, 则将2只小鼠的视网膜色素上皮细胞从12毫米聚酯膜插入预加载到12井培养板中。电镀细胞高密度是需要的, 因为在这种情况下, RPE 细胞能够保持原有的性质, 如六角形和色素沉着。</li>
	</ol>
</li>
	<li>不要移动板块或改变培养前72小时的生长培养基。3天后, 用新鲜的预热生长培养基取代旧培养基。以后那改变文化媒介每隔一天。一旦视网膜色素细胞得到融合, 将生长培养基中的血清减少到2%。 注: 细胞可以使用0.25% 胰蛋白酶分裂。然而, 分裂后的细胞可能会失去其六角形和色素沉着在随后的段落。我们不建议分裂细胞, 但如果它是需要的下游应用的文化, 请记住, 主要细胞不能无限期传代: 他们被称为消除区分后 5-7 通道。</li>
</ol>

<ol>
	<li>Mart&#237;nez-Morales, J. R., Rodrigo, I., Bovolenta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+development:+a+view+from+the+retina+pigmented+epithelium.">Eye development: a view from the retina pigmented epithelium.</a> Bioessays. 26, 766-777 (2004).</li><li>Lehmann, G. L., Benedicto, I., Philp, N. J., Rodriguez-Boulan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+protein+polarity+and+trafficking+in+RPE+cells:+past,+present+and+future.">Plasma membrane protein polarity and trafficking in RPE cells: past, present and future.</a> Exp Eye Res. 126, 5-15 (2014).</li><li>Fronk, A. H., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+retinal+pigment+epithelial+cells:+a+review+of+current+protocols+and+future+recommendations.">Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations.</a> J Tissue Eng. 7, (2016).</li><li>Rizzolo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+properties+of+cultured+retinal+pigment+epithelium.">Barrier properties of cultured retinal pigment epithelium.</a> Exp Eye Res. 126, 16-26 (2014).</li><li>Lu, F., Yan, D., Zhou, X., Hu, D. N., Qu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+melanin-related+genes+in+cultured+adult+human+retinal+pigment+epithelium+and+uveal+melanoma+cells.">Expression of melanin-related genes in cultured adult human retinal pigment epithelium and uveal melanoma cells.</a> Mol Vis. 13, 2066-2072 (2007).</li><li>Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+melanin+and+lipofuscin+in+RPE+cell+culture+models.">Studying melanin and lipofuscin in RPE cell culture models.</a> Exp Eye Res. 126, 61-67 (2014).</li><li>Saini, J. S., Temple, S., Stern, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Retinal+Pigment+Epithelium+Stem+Cell+(RPESC).">Human Retinal Pigment Epithelium Stem Cell (RPESC).</a> Adv Exp Med Biol. 854, 557-562 (2016).</li><li>Akrami, H., 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=Retinal+pigment+epithelium+culture;a+potential+source+of+retinal+stem+cells.">Retinal pigment epithelium culture;a potential source of retinal stem cells.</a> J Ophthalmic Vis Res. 4, 134-141 (2009).</li><li>Hu, J., Bok, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+cultured+human+fetal+retinal+pigment+epithelium+in+studies+of+the+classical+retinoid+visual+cycle+and+retinoid-based+disease+processes.">The use of cultured human fetal retinal pigment epithelium in studies of the classical retinoid visual cycle and retinoid-based disease processes.</a> Exp Eye Res. , 46-50 (2014).</li><li>Mazzoni, F., Safa, H., Finnemann, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+photoreceptor+outer+segment+phagocytosis:+use+and+utility+of+RPE+cells+in+culture.">Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture.</a> Exp Eye Res. 126, 51-60 (2014).</li><li>Reichhart, N., Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+and+transporters+of+the+retinal+pigment+epithelium.">Ion channels and transporters of the retinal pigment epithelium.</a> Exp Eye Res. 126, 27-37 (2014).</li><li>Valapala, 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=Lysosomal-mediated+waste+clearance+in+retinal+pigment+epithelial+cells+is+regulated+by+CRYBA1/&#946;A3/A1-crystallin+via+V-ATPase-MTORC1+signaling.">Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/&#946;A3/A1-crystallin via V-ATPase-MTORC1 signaling.</a> Autophagy. 10, 480-496 (2014).</li><li>Shang, 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=The+amino+acid+transporter+SLC36A4+regulates+the+amino+acid+pool+in+retinal+pigmented+epithelial+cells+and+mediates+the+mechanistic+target+of+rapamycin,+complex+1+signaling.">The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.</a> Aging Cell. , (2017).</li><li>Fernandez-Godino, R., Garland, D. L., Pierce, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+characterization+of+primary+mouse+RPE+cells.">Isolation, culture and characterization of primary mouse RPE cells.</a> Nat Protoc. 11, 1206-1218 (2016).</li><li>Pfeffer, B. A., Philp, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+retinal+pigment+epithelium:+Special+Issue.">Cell culture of retinal pigment epithelium: Special Issue.</a> Exp Eye Res. 126, 1-4 (2014).</li><li>Singh, S., Woerly, S., McLaughlin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+artificial+substrates+for+retinal+pigment+epithelial+monolayer+transplantation.">Natural and artificial substrates for retinal pigment epithelial monolayer transplantation.</a> Biomaterials. 22, 3337-3343 (2001).</li><li>Sonoda, 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=A+protocol+for+the+culture+and+differentiation+of+highly+polarized+human+retinal+pigment+epithelial+cells.">A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.</a> Nat Protoc. 4, 662-673 (2009).</li><li>Ho, T. C., Del Priore, L. V., Kaplan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+of+retinal+pigment+epithelium+following+isolation+with+a+gelatin+matrix+technique.">Tissue culture of retinal pigment epithelium following isolation with a gelatin matrix technique.</a> Experimental eye research. 64, 133-139 (1997).</li><li>Toops, K. A., Tan, L. X., Lakkaraju, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+three-step+protocol+for+live+imaging+of+intracellular+traffic+in+polarized+primary+porcine+RPE+monolayers.">A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers.</a> Exp Eye Res. , 74-85 (2014).</li><li>Gibbs, D., Kitamoto, J., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+phagocytosis+by+retinal+pigmented+epithelium+that+lacks+myosin+VIIa,+the+Usher+syndrome+1B+protein.">Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein.</a> Proc Natl Acad Sci USA. 100, 6481-6486 (2003).</li><li>Gibbs, D., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+mouse+retinal+pigmented+epithelial+cells.">Isolation and culture of primary mouse retinal pigmented epithelial cells.</a> Adv Exp Med Biol. 533, 347-352 (2003).</li><li>Pitk&#228;nen, L., Ranta, V. P., Moilanen, H., Urtti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+retinal+pigment+epithelium:+effects+of+permeant+molecular+weight+and+lipophilicity.">Permeability of retinal pigment epithelium: effects of permeant molecular weight and lipophilicity.</a> Invest Ophthalmol Vis Sci. 46, 641-646 (2005).</li><li>Mao, Y., Finnemann, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+photoreceptor+outer+segment+phagocytosis+by+RPE+cells+in+culture.">Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture.</a> Methods Mol Biol. 935, 285-295 (2013).</li></ol>

DS 已获得拜耳医疗保健, 德国和 f. 霍夫曼-罗氏, 瑞士的研究资金。其余的作者声明没有竞争的财政利益。这项工作是支持的研究, 以防止失明 (不受限制的赠款威尔默眼科研究所和匹兹堡大学)。 这项工作还由匹兹堡大学眼科的初创基金支持。

所提出的详细协议能够可靠地建立在1周后到达融合的小鼠原发性视网膜色素培养物, 并在2周后呈现主要的视网膜色素特征, 如六角形和色素沉着。所获得的 RPE 细胞可用于一些下游应用, 如维生素 a 代谢9, 感光外段的吞噬功能10和离子传输11, 上皮细胞极性2, 溶酶体稳态和自噬12,13。根据下游应用程序的不同, 派生区域性的形态学可以通过传输和扫描电子显微镜进行验证, 如14中详细描述的那样。额外的化验, 以证明成熟的文化可能包括 transepithelial 电阻 (志愿者) 测试, 以显示视网膜色素上皮细胞的极性, 渗透性检测4,22, 功能测试 (吞噬23) 和圆顶形成。
在我们的经验中, 在井中镀的细胞越多, 融合和表型越好。在本协议中, 我们将2只小鼠的 RPE 细胞从12毫米的插入物中播种。然而, 将视网膜色素上皮细胞从2只小鼠植入6.5 毫米插入物, 以获得更好的融合和志愿者也是可行的。在许多研究中, 视网膜色素上皮细胞培养皿上涂有胶或层粘连蛋白。我们没有看到在视网膜色素细胞附着在这些血管有或没有涂层的显著差异。此外, 可以对培养基中的血清中的百分比进行修改, 以取得更好的效果。10% 的血清通常有利于达到融合, 但下降到较少的血清似乎有利于视网膜色素分化/成熟14,17。
这种方法的主要局限性是需要对进行眼部解剖的人进行精确的训练, 以确保视网膜色素上皮细胞恢复的最高效率, 而不会与其他眼部组织的细胞交叉污染。为了避免交叉污染, 研究者应该掌握这个过程的关键步骤: 仔细剥离视网膜, 然后从脉络膜中精确分离视网膜色素。获得的文化纯度可以使用 RPE 特定的染色来验证。许多 rpe 标记可用于描述视网膜色素上皮的培养, 包括视觉周期、屏障和传输功能、新陈代谢、吞噬能力和黑15的相关基因。
另一个限制是可以同时处理的眼睛数量, 以获得文化。一般而言, 我们不建议一次使用2只老鼠。为了确保视网膜色素上皮细胞的活力, 应尽快解剖眼睛。当用户变得更精通, 有可能处理更多的老鼠通过交替孵化时间。
总的来说, 在建立主要的鼠标 RPE 区域性3、14、15等方面已经发布了许多方法, 但是, 我们第一次提出完整协议的视频版本。本协议可作为眼科解剖技术的指导。然而, 最好的平衡之间的高速和精度的解剖所需的, 只有在实践中才能实现。

视网膜色素上皮 (RPE) 是一个单一层的极化上皮细胞位于神经视网膜和脉络膜之间的眼睛。视网膜色素上皮细胞的功能和 rpe 单层的完整性对视力是至关重要的, 因为视网膜色素上皮在维持外血-视网膜屏障、水和离子在视网膜之间的传输等多个过程中起着重要作用。脉络膜, 光吸收, 保护免受氧化应激, 控制甲酸代谢, 和吞噬的外部部分的感光细胞1,2。视网膜色素在眼睛后的位置, 以及它的屏障功能, 防止药物管理系统地从血液传递到玻璃体的幽默, 使人们很难研究视网膜色素功能在体内的复杂性。因此, 很有必要建立视网膜色素细胞培养, 以研究在一个灵活的, 受控环境中的 rpe 细胞3,4。
有许多已建立的 RPE 细胞线存在, 提供了一种方便和方便的方式获取和储存的细胞;但是, 与主单元格2、3、4相比, 传代单元格有一些缺点。首先, 它们通常以细胞形态学的变化为特征。例如, 由于细胞极性表型的丧失和紧连接的部分消失4, 现有的细胞系都没有发现适合于对 RPE 屏障特性进行可靠的研究。除了极性的丧失和细胞对细胞的正常连接外, 视网膜色素上皮细胞系由于在成人视网膜色素沉着5中没有关键的黑酶而迅速失去色素。色素沉着可以恢复, 但对重新着色机制的综合分析, 将包括透射电镜, 基因表达分析和化学化验的结合, 以确认黑色素的存在从来没有已执行6。还有一个限制是视网膜色素上皮细胞系延长了细胞的寿命 (有时是不朽的), 在某些条件下, 可以转化为自更新的多能干细胞, 分离出基质, 形成漂浮的菌落7,8. 这一限制使得无法使用细胞系进行移植实验3。
考虑到已建立的视网膜色素细胞系的缺点, 从新鲜组织获得的原发性视网膜色素上皮细胞培养可能成为研究视网膜色素的一种更具生物学意义的模型。主要的视网膜色素细胞不仅用于研究视网膜色素特异功能, 如维生素 A 代谢9, 感光外段的吞噬作用10和离子传输11, 但也研究基本细胞生物学, 如上皮细胞极性2 , 溶酶体稳态和自噬12,13。
在过去的几年中, 有许多关于建立主要视网膜色素培养的出版物, 表明对这一研究领域的兴趣越来越浓厚3,14,15。许多人类视网膜色素细胞和非人类视网膜色素上皮细胞的协议, 如牛和猪视网膜色素上皮细胞被发布了16,17,18,19。然而, 由于它们的体积小得多, 所以处理小鼠视网膜色素细胞更加困难。尽管相当多的出版物已经描述了将 rpe 细胞从鼠标14、20、21中隔离出来的协议, 但仍有许多研究人员在努力隔离 rpe 细胞而不神经视网膜碎片对脉络膜细胞或细胞的污染。在这里, 我们提出了建立主要的小鼠 RPE 细胞培养的协议, 包括从老鼠的眼睛, 眼睛的解剖和视网膜色素表的分离, 以获得细胞培养。该视频协议将特别有用的研究员谁开始工作的小鼠主要视网膜色素培养和需要指导的解剖技术。

<table border="0" cellpadding="0" cellspacing="0" width="980">
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			<td style="width:123px;">500260</td>
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			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Syringe, 5 mL</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Inverted Laboratory Microscope&#160;Leica DM IL LED</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipette</td>
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			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Barrier and non-filtered pipette tips</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

primary cell cultures from the mouse retinal pigment epithelium

视网膜色素上皮 (RPE) 是一种高度极化的多功能上皮, 位于神经视网膜和眼脉络之间。这是一个单一的色素细胞, 是六边形包装和连接紧密交界处。视网膜色素的主要功能包括光的吸收、棚感光外段的吞噬、离子的空间缓冲、养分的输送、离子和水以及在视觉循环中的积极参与。对视网膜色素上皮细胞的生物学研究具有重要而多样的作用。已经建立了一些视网膜色素上皮细胞系;然而, 传代和永生化细胞很快就失去了自然视网膜色素细胞的一些形态学和生理特性。因此, 主细胞更适合研究视网膜色素上皮细胞生物学和功能的不同方面。小鼠原发性视网膜色素上皮细胞培养对研究人员非常有用, 因为小鼠模型在生物研究中被广泛应用, 然而从老鼠那里采集视网膜色素细胞也很有挑战性, 因为它们的体积小。在这里, 我们提出了建立主要的小鼠 RPE 细胞培养的协议, 包括眼睛的摘除和解剖和视网膜色素表的分离, 以产生细胞培养。此方法可实现有效的单元恢复。从两个小鼠获得的 RPE 细胞可以达到融合在一个12毫米聚酯膜插入预加载的文化板块后, 一个星期的文化, 并显示一些原始的性质, 如六角形和色素沉着在两个周的文化。

2只小鼠在12毫米聚酯膜插入物中建立的小鼠原发性视网膜色素培养在1周的培养后达到 90-95% 融合。经过2周的培养, 细胞达到100% 融合, 并开始形成一个镶嵌的六角, 有色和双核细胞。3周的文化, 细胞继续形成的形状和色素沉着, 然而, 4 周后, 部分细胞得到了 hyperpigmented (图 2)。
主要视网膜色素细胞培养的纯度可以通过染色使用 RPE65 抗体 (甲酸 isomerohydrolase, 这是脊椎动物视觉循环中的关键酶, 使所有反式视黄基酯转化为 11-顺黄醇, 在phototransduction) (图 3, 红色)。通过 phalloinin 染色 (图 3, 绿色) 可以证明细胞对细胞连接的完整性和六边形形状的存在。此外, ZO-1 蛋白的表达, 紧密连接的重要组成部分, 可以验证使用西方印迹 (图 4) 或染色。
结果表明, 所获得的小鼠主要视网膜色素培养能够增殖, 保持色素沉着, 六角形和紧密连接, 以及表达 RPE65 等功能标志物。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56997/56997fig1.jpg"> 图 1.不同的眼睛解剖步骤, 以获得主要视网膜色素细胞培养.(a) 在2% 酶分离后, 眼睛在生长培养基中冲洗。(b)通过移除角膜和晶状体, 获得后眼罩。(c)通过移除相关的虹膜色素上皮, 进一步清除由此产生的眼罩。(d)在生长培养基进一步孵化后, 眼罩被径向切开以产生象限。(e)神经视网膜从其余的 RPE-脉络膜-巩膜复合体剥离。(f)视网膜色素表从刷膜中轻轻地分离出来。注意, 视网膜色素上皮看起来更褐色 (箭头), 而脉络膜更暗, 非常粘稠。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56997/56997fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56997/56997fig2.jpg"> 图 2.3周大鼠在12毫米聚酯膜上培养的主要视网膜色素细胞, 在12井培养板上预加载.2只小鼠在12毫米聚酯膜插入物中建立的小鼠原发性视网膜色素培养在1周的培养 (a) 后达到 90-95% 融合。经过2周的培养, 细胞达到100% 融合, 并开始形成一个镶嵌的六角, 有色和双核细胞 (b)。经过3周的培养, 细胞继续形成形状和色素沉着 (c), 然而, 在4周后, 部分细胞得到 hyperpigmented (d)。刻度条: 100 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56997/56997fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56997/56997fig3.jpg"> 图 3.视网膜色素上皮细胞标记, RPE65, 是可检测到的主要小鼠视网膜色素细胞.在4周的培养后, 主要的小鼠视网膜色素细胞固定在4% 多聚甲醛 (粉煤灰) 中, 并以 RPE65 抗体或阻断缓冲作为阴性对照 (NC) 孵化。罗丹明 (绿色) 和 DAPI (蓝色) 也被添加来染色细胞膜和细胞核。刻度条: 100 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56997/56997fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56997/56997fig4.jpg"> 图 4.紧密连接的标记, ZO-1 在主要视网膜色素细胞中表达.主要的小鼠视网膜色素细胞 (mRPE) 是在3周的培养和8µg 的细胞裂解被运行在一个西方污点。ARPE-19 细胞的等效量 (商业上可用的稳定 RPE 细胞系) 被用作阴性对照。ZO-1 在主要视网膜色素细胞中被高度表达, 而 ARPE-19 细胞缺乏。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56997/56997fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

视网膜色素上皮 (RPE) 是一种多功能的眼睛上皮。在这里, 我们提出了建立从小鼠视网膜色素形成的主要细胞培养的协议。

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 ...

primary-cell-cultures-from-the-mouse-retinal-pigment-epithelium

Nanyang Technological University

Research

JoVE Journal

Biology

20.9K 次观看.  University of Pittsburgh School of Medicine. 视网膜色素上皮 (RPE) 是一种多功能的眼睛上皮。在这里, 我们提出了建立从小鼠视网膜色素形成的主要细胞培养的协议。

视频: Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

科研

生物学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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, ...

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 ...