Name: isolation of primary mouse retinal pigmented epithelium cells
Uploaded: 2022-11-04T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Primary Mouse Retinal Pigmented Epithelium Cells at JoVE.com

这项工作得到了国家眼科研究所（NEI），国家眼科研究所（NEI）基金R01 EY029751-04的支持。

根据奥克兰大学IACUC动物协议编号21063的指南和ARVO在眼科和视觉研究中使用动物的声明的指南使用动物。
1. 溶液制备
<ol><li>通过补充 Dulbecco 的改良鹰培养基/营养混合物 F-12 （DMEM/F12） 和 25% 胎牛血清 （FBS）、1.5% 青霉素/链霉素和 0.02% 庆大霉素来制备完整的 RPE 细胞培养基。</li>
	<li>通过向 741 μL Hank 平衡盐溶液 （HBSS） 补充 127 μL 胶原酶储备溶液和 132 μL 透明?...

<ol>
	<li>Young, R. W., Droz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+renewal+of+protein+in+retinal+rods+and+cones.">The renewal of protein in retinal rods and cones.</a> The Journal of Cell Biology. 39 (1), 169-184 (1968).</li><li>Sparrow, J. R., Hicks, D., Hamel, C. P. <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+in+health+and+disease.">The retinal pigment epithelium in health and disease.</a> Current Molecular Medicine. 10 (9), 802-823 (2010).</li><li>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=The+retinal+pigment+epithelium+in+visual+function.">The retinal pigment epithelium in visual function.</a> Physiological Reviews. 85 (3), 845-881 (2005).</li><li>Marlhens, F., 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=Autosomal+recessive+retinal+dystrophy+associated+with+two+novel+mutations+in+the+RPE65+gene.">Autosomal recessive retinal dystrophy associated with two novel mutations in the RPE65 gene.</a> European Journal of Human Genetics. 6 (5), 527-531 (1998).</li><li>Morimura, 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=Mutations+in+the+RPE65+gene+in+patients+with+autosomal+recessive+retinitis+pigmentosa+or+leber+congenital+amaurosis.">Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis.</a> Proceedings of the National Academy of Sciences. 95 (6), 3088-3093 (1998).</li><li>Weiter, J. J., Delori, F. C., Wing, G. L., Fitch, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+pigment+epithelial+lipofuscin+and+melanin+and+choroidal+melanin+in+human+eyes.">Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes.</a> Investigative Ophthalmology &amp; Visual Science. 27 (2), 145-152 (1986).</li><li>Feeney-Burns, L., Hilderbrand, E. S., Eldridge, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+human+RPE:+morphometric+analysis+of+macular,+equatorial,+and+peripheral+cells.">Aging human RPE: morphometric analysis of macular, equatorial, and peripheral cells.</a> Investigative Ophthalmology &amp; Visual Science. 25 (2), 195-200 (1984).</li><li>Ibrahim, A. 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=Hyperhomocysteinemia+disrupts+retinal+pigment+epithelial+structure+and+function+with+features+of+age-related+macular+degeneration.">Hyperhomocysteinemia disrupts retinal pigment epithelial structure and function with features of age-related macular degeneration.</a> Oncotarget. 7 (8), 8532-8545 (2016).</li><li>Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration:+review+of+pathogenesis.">Age-related macular degeneration: review of pathogenesis.</a> European Journal of Ophthalmology. 8 (4), 199-206 (1998).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARPE-19,+a+human+retinal+pigment+epithelial+cell+line+with+differentiated+properties.">ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.</a> Experimental Eye Research. 62 (2), 155-170 (1996).</li><li>Flannery, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+animal+models+for+the+study+of+inherited+retinal+dystrophies.">Transgenic animal models for the study of inherited retinal dystrophies.</a> ILAR Journal. 40 (2), 51-58 (1999).</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> Advances in Experimental Medicine and Biology. 533, 347-352 (2003).</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> Nature Protocols. 11 (7), 1206-1218 (2016).</li><li>Samra, Y. 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=Implication+of+N-Methyl-d-Aspartate+receptor+in+homocysteine-induced+age-related+macular+degeneration.">Implication of N-Methyl-d-Aspartate receptor in homocysteine-induced age-related macular degeneration.</a> International Journal of Molecular Sciences. 22 (17), 9356 (2021).</li><li>van Marum, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+use+of+memantine+in+Alzheimer's+disease.">Update on the use of memantine in Alzheimer's disease.</a> Neuropsychiatric Disease and Treatment. 5, 237-247 (2009).</li><li>Elsherbiny, N. 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=Homocysteine+induces+inflammation+in+retina+and+brain.">Homocysteine induces inflammation in retina and brain.</a> Biomolecules. 10 (3), 393 (2020).</li><li>Tawfik, A., Elsherbiny, N. M., Zaidi, Y., Rajpurohit, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homocysteine+and+age-related+central+nervous+system+diseases:+role+of+inflammation.">Homocysteine and age-related central nervous system diseases: role of inflammation.</a> International Journal of Molecular Sciences. 22 (12), 6259 (2021).</li><li>Shang, P., Stepicheva, N. A., Hose, S., Zigler Jr, J. S., Sinha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cell+cultures+from+the+mouse+retinal+pigment+epithelium.">Primary cell cultures from the mouse retinal pigment epithelium.</a> Journal of Visualized Experiments. (133), e56997 (2018).</li><li>Chen, 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=Characterization+of+a+spontaneous+mouse+retinal+pigment+epithelial+cell+line+B6-RPE07.">Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07.</a> Investigative Ophthalmology &amp; Visual Science. 49 (8), 3699-3706 (2008).</li><li>AVMA Guidelines for the Euthanasia of Animals: 2020 Edition. Available from: <a target="_blank" href="https://www.avma.org/KB/Policies/Documents/euthanasia.pdf">https://www.avma.org/KB/Policies/Documents/euthanasia.pdf</a> (2020)</li><li>Cai, X., Conley, S. M., Naash, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPE65:+role+in+the+visual+cycle,+human+retinal+disease,+and+gene+therapy.">RPE65: role in the visual cycle, human retinal disease, and gene therapy.</a> Ophthalmic Genetics. 30 (2), 57-62 (2009).</li><li>P&#233;rez-&#193;lvarez, M. 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=Vimentin+isoform+expression+in+the+human+retina+characterized+with+the+monoclonal+antibody+3CB2.">Vimentin isoform expression in the human retina characterized with the monoclonal antibody 3CB2.</a> Journal of Neuroscience Research. 86 (8), 1871-1883 (2008).</li><li>Tawfik, A., Samra, Y. A., Elsherbiny, N. M., Al-Shabrawey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+hyperhomocysteinemia+in+blood+retinal+barrier+(BRB)+dysfunction.">Implication of hyperhomocysteinemia in blood retinal barrier (BRB) dysfunction.</a> Biomolecules. 10 (8), 1119 (2020).</li><li>Promsote, W., Makala, L., Li, B., Smith, S. B., Singh, N., Ganapathy, V., Pace, B. S., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monomethylfumarate+induces+&#947;-globin+expression+and+fetal+hemoglobin+production+in+cultured+human+retinal+pigment+epithelial+(RPE)+and+erythroid+cells,+and+in+intact+retina.">Monomethylfumarate induces &#947;-globin expression and fetal hemoglobin production in cultured human retinal pigment epithelial (RPE) and erythroid cells, and in intact retina.</a> Invest Ophthalmol Vis Sci. 55 (8), 5382-5393 (2014).</li></ol>

当前协议是从小鼠眼睛中分离RPE的报告，修改和简化的详细程序。该协议包括从小鼠眼睛分离的RPE细胞的剜除，解剖，收集，接种，培养和表征。
成功进行RPE分离必须满足一些限制和关键步骤，例如小鼠年龄，解剖的眼睛数量，组织培养板或培养皿的大小以及接种，储存和传代后的注意事项。为了能够传代分离的细胞多达三到四次，最好的小鼠年龄在18-21天之间。为了获得合...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63543">Isolation of Primary Mouse Retinal Pigmented Epithelium Cells</a>. The Discussion and References sections were updated.
The third paragraph of the Discussion&#160;section was updated from:
This protocol is different from other valuable and reliable published protocols for primary mouse RPE isolation13,19,20 in that it is simplified with a few steps that are easy to follow. RPE cells are able to be identified by their morphology, pigmentation, and specific RPE markers such as RPE654,5,21. Validation of the purity and contamination of M&#252;ller cells was achieved by staining isolated cells with a specific cell marker for M&#252;ller cells (vimentin)22. Many techniques have been used to evaluate the integrity of the blood-retinal barrier (BRB) in vitro, such as electron microscope (EM) examination, assessment of tight junction proteins (TJPs), FITIC dextran leakage assay, and transepithelial electrical resistance (TER) measurement that evaluates the physiological function of RPE as a monolayer8,23. RPE cells play an important role in maintaining the outer BRB, and to validate this function, we evaluated tight junction proteins (TJPs) such as Zona ocludin-1 (ZO-1) and cytoskeletal proteins like F-actin as previously published8. TJPs evaluation is a more convenient method for the evaluation of BRB integrity and barrier function which doesn&#39;t require expensive equipment that may not be available in all research labs, as opposed to EM evaluation or TER.
to:
This protocol is different from other valuable and reliable published protocols for primary mouse RPE isolation13,19,20,24 in that it is simplified with a few steps that are easy to follow. RPE cells are able to be identified by their morphology, pigmentation, and specific RPE markers such as RPE654,5,21. Validation of the purity and contamination of M&#252;ller cells was achieved by staining isolated cells with a specific cell marker for M&#252;ller cells (vimentin)22. Many techniques have been used to evaluate the integrity of the blood-retinal barrier (BRB) in vitro, such as electron microscope (EM) examination, assessment of tight junction proteins (TJPs), FITIC dextran leakage assay, and transepithelial electrical resistance (TER) measurement that evaluates the physiological function of RPE as a monolayer8,23. RPE cells play an important role in maintaining the outer BRB, and to validate this function, we evaluated tight junction proteins (TJPs) such as Zona ocludin-1 (ZO-1) and cytoskeletal proteins like F-actin as previously published8. TJPs evaluation is a more convenient method for the evaluation of BRB integrity and barrier function which doesn&#39;t require expensive equipment that may not be available in all research labs, as opposed to EM evaluation or TER.
In the References section, a 24th reference was added:
<ol start="24">
	<li>Promsote, W., Makala, L., Li, B., Smith, SB., Singh, N., Ganapathy, V., Pace, B.S., Martin, P. Monomethylfumarate induces &#947;-globin expression and fetal hemoglobin production in cultured human retinal pigment epithelial (RPE) and erythroid cells, and in intact retina. Invest Ophthalmol Vis Sci. 55(8):5382-93 (2014).</li>
</ol>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63543">Isolation of Primary Mouse Retinal Pigmented Epithelium Cells</a>. The Discussion and References sections were updated.

Erratum: Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

视网膜色素上皮（RPE）细胞位于脉络膜和神经视网膜之间，形成位于感光器（PR）细胞后面的简单单层立方体细胞1。RPE在维持PR细胞的健康环境中起着关键作用，主要是通过减少活性氧（ROS）的过度积累和随之而来的氧化损伤1。RPE细胞监督许多功能，例如类视黄醇的转化和储存，散射光的吸收，液体和离子运输以及脱落的PR外段膜2，3的吞噬作用。RPE（形态/功能）的改变会损害其功能，导致视网膜病变，这是许多眼部疾病的共同特征4。许多眼部疾病与RPE细胞形态和功能的改变有关，包括一些遗传性疾病，如色素性视网膜炎、Leber先天性黑朦和白化病4，5，6，以及与年龄相关的眼部疾病，如糖尿病视网膜病变（DR）和年龄相关性黄斑变性（AMD）7，8.人类细胞是最理想的，因此研究原代人RPE细胞中的RPE疾病以形成RPE单层是理想的。然而，伦理问题和人类供体的可用性有限，因为大多数这些疾病导致发病率9，但不一定导致死亡率10，从而阻止了原代人RPE细胞的分离。这使得培养来自非人类动物供体的RPE细胞成为首选的替代方案。啮齿动物，特别是小鼠，被认为是研究不同眼部疾病的绝佳模型，因为转基因技术在这些物种中得到了更广泛的建立11。尽管使用培养的原代RPE细胞具有许多优点，但很难维持生长的细胞进行多次传代，或者储存和重复使用细胞。该协议的主要限制是小鼠的年龄;用于RPE分离的小鼠应该非常年轻（18-21天大是最佳的），因为很难从成年小鼠11，12，13培养RPE细胞。RPE细胞可以在任何年龄从小鼠眼睛中分离出来，但是多达四次细胞传代仅在年轻小鼠（18-21天大）中成功。使用C57BL6小鼠和在RPE细胞上缺失N-甲基-D-天冬氨酸受体（NMDARs）的转基因小鼠从小鼠视网膜中分离RPE以研究升高的氨基酸同型半胱氨酸对AMD14发育和进展的影响。此外，分离的原代RPE细胞通过抑制RPE细胞的NMDAR来帮助提出AMD的治疗靶点14。有一些NMDAR阻滞剂已获得美国食品和药物管理局（FDA）的批准，目前用于治疗与阿尔茨海默病（AD）相关的中度至重度混乱（痴呆），例如美金刚16，这可能是AMD14的潜在治疗靶点。此外，分离的原代小鼠RPE细胞用于检测炎症标志物，并使用转基因小鼠（CBS）诱导炎症作为同型半胱氨酸诱导的AMD和AD特征的潜在机制，其表现出高水平的同型半胱氨酸16，17。
该方案用于从我们实验室开发的野生型C57BL / 6小鼠和转基因小鼠中分离RPE细胞，作为其他已发布的分离方案13，18，19的简化改编，以达到易于应用和可靠的方案。该协议中没有性别偏好。虽然小鼠年龄对分离过程至关重要，但年轻，年龄的小鼠（18-21天大）和任何年龄（长达12个月）的老年小鼠用于RPE分离。然而，我们注意到从年轻小鼠中分离的RPE细胞寿命更长，最多可以进行四次传代。从老年小鼠中分离的RPE细胞可以传代一次或两次，然后它们将停止以正常速率生长并改变其形状以更细长（成纤维细胞样细胞）。还观察到色素沉着丧失和对组织培养板的粘附降低，随后出现脱落。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beaker : 100mL</td>
			<td>KIMAX</td>
			<td>14000</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C7657-25MG</td>
			<td>For working enzyme, A</td>
		</tr>
		<tr>
			<td>Disposable Graduated Transfer Pipettes :3.2mL Sterile</td>
			<td></td>
			<td>13-711-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>&#160;gibco&#160;</td>
			<td>11330</td>
			<td>Media to grow RPE cells&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>gibco</td>
			<td>26140079</td>
			<td>For complete RPE cell culture media</td>
		</tr>
		<tr>
			<td>Gentamicin Reagent Solution</td>
			<td>gibco</td>
			<td>15750-060</td>
			<td>For complete RPE cell culture media</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Thermo Scientific</td>
			<td>88284</td>
			<td>For working enzymes (A&#38;B)&#160;</td>
		</tr>
		<tr>
			<td>Heracell VISO 160i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase from bovine testes</td>
			<td>Sigma-Aldrich</td>
			<td>H3506-500MG</td>
			<td>For working enzyme A</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-Lok Syringe with attached needle 21 G x 1 1/2 in., sterile, single use, 3 mL</td>
			<td>B-D</td>
			<td>309577</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge Tube: 2 mL</td>
			<td>Grainger</td>
			<td>11L819</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-RPE65 antibody&#160;</td>
			<td>Abcam, Cambridge, MA, USA</td>
			<td>ab78036</td>
			<td>For IF staining&#160;</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>gibco</td>
			<td>15140-122</td>
			<td>For complete RPE cell culture media</td>
		</tr>
		<tr>
			<td>Positive Action Tweezers, Style 5/45</td>
			<td>Dumont</td>
			<td>72703-DZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors Iris Standard Straight 11.5cm</td>
			<td>GARANA INDUSTRIES</td>
			<td>2595</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall St8 Centrifuge</td>
			<td>ThermoScientific</td>
			<td>75007200</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 305 Microscope</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade, #11, Stainless Steel</td>
			<td>Bard-Parker</td>
			<td>371211</td>
			<td></td>
		</tr>
		<tr>
			<td>Suspension Culture Dish 60mm x 15mm Style</td>
			<td>Corning</td>
			<td>430589</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish : 100x20mm style</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tornado Tubes: 15mL</td>
			<td>Midsci</td>
			<td>C15B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tornado Tubes: 50mL</td>
			<td>Midsci</td>
			<td>C50R</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA (1x) 0.25%</td>
			<td>gibco</td>
			<td>2186962</td>
			<td>For working enzyme B</td>
		</tr>
		<tr>
			<td>Tweezers 5MS, 8.2cm, Straight, 0.09x0.05mm Tips</td>
			<td>Dumont</td>
			<td>501764</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers Positive Action Style 5, Biological, Dumostar, Polished Finish, 110 mm OAL</td>
			<td>Electron Microscopy Sciences Dumont</td>
			<td>50-241-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Underpads, Moderate : 23&#34; X 36&#34;</td>
			<td>McKesson</td>
			<td>4033</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 2.5mm Cutting Edge</td>
			<td>FST</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss AxioImager Z2</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Zen Blue 2.6</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary mouse retinal pigmented epithelium cells

视网膜色素上皮（RPE）层紧邻光感受器后面，并拥有一个复杂的代谢系统，在维持光感受器的功能中起着几个关键作用。因此，RPE结构和功能对于维持正常视力至关重要。该手稿介绍了原代小鼠RPE细胞分离的既定方案。RPE分离是研究不同眼部疾病小鼠模型中RPE病理学分子机制的好工具。此外，RPE分离可以帮助比较从野生型和转基因小鼠中分离的原代小鼠RPE细胞，以及测试可以加速视觉障碍治疗开发的药物。手稿提出了一个循序渐进的RPE隔离方案;整个过程，从眼球摘除到播种，大约需要4个小时。接种后5-7天内不应更换培养基，以使分离的细胞不受干扰地生长。该过程之后通过免疫荧光 表征 细胞中的形态、色素沉着和特异性标志物。细胞最多可以传代三到四次。

验证分离的 RPE 细胞的特异性、纯度和屏障功能/形成 在光学显微镜下检查分离的细胞以验证其活力，形态和色素沉着。捕获来自P0和P1的图像（图1A，B）以及来自P0和P4的图像（图1C，D）以显示细胞的变化;当传代进行到第四代时，形状，大小和色素沉着（黑色箭头指向P4中分离的RPE细胞）。RPE65蛋白的抗体用于验证分离细胞的?...

Watch this Scientific Journal Video about Isolation of Primary Mouse Retinal Pigmented Epithelium Cells at JoVE.com

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

本手稿描述了一种逐步从小鼠眼睛中分离视网膜色素上皮（RPE）细胞的简化方案。该方案包括小鼠眼睛的剜除和解剖，然后是RPE细胞的分离，接种和培养。

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 ...

该协议将概述一种成功分离原代小鼠视网膜色素上皮细胞的简单有效的方法。视网膜色素上皮细胞在维持感光细胞和保证视网膜的正常功能方面起着非常重要的作用。我们的实验室一直在分离小鼠原代色素上皮细胞，作为研究外血视网膜屏障和相关疾病（如年龄相关性黄斑变性）的重要工具。要提取小鼠眼睛，请根据您所在机构批准的方法对小鼠实施道德安乐死，然后将鼠标放在无菌垫上，并通过在眼眶区域按压 5/45 型镊子来诱导眼球突出，然后将镊子移动到眼睛的后部并轻轻拉动以移除眼睛，确保视神经仍然完好无损。使用镊子时，将眼睛浸入含有5%聚维酮碘的2毫升微量离心管中。在室温下孵育眼睛 2 至 3 分钟。从聚维酮碘中取出眼睛，并将它们放入培养皿中。使用无菌转移移液器时，用汉克斯平衡盐水溶液清洗眼睛，直到聚维酮碘的橙色消失。这大约需要 2 到 3 次洗涤。将眼睛放在新的培养皿中，并将其浸没在冷完全RPE细胞生长培养基中。从那里，您可以在解剖显微镜下开始解剖。在显微镜下，使用镊子和/或Vannas剪刀拉开或切掉眼外肌中的结缔组织。然后可以在冷的RPE细胞培养基中保持眼睛一小时。但是，最好在清洁眼球后直接进行下一步。将眼睛转移到含有酶A溶液的2毫升微量离心管中，然后将眼睛在37摄氏度的培养箱内孵育40分钟。从酶A溶液中取出眼睛，并将它们放入含有酶B溶液的新2毫升微量离心管中，然后将它们放入培养箱中并在37摄氏度下孵育50分钟。从酶B中取出眼睛，将它们放入60毫米培养皿中，浸没并完成RPE细胞生长培养基，然后将眼睛在4摄氏度下孵育30分钟。将培养皿放在解剖显微镜下并开始解剖。使用M5S镊子和注射器针头在ora seratta部分做一个切口。现在用两个镊子，用一个镊子抓住切口的内部，用另一个镊子抓住角膜。通过拉角膜开始慢慢地从视网膜上撕裂角膜。一定要以小的增量撕裂，并在去除角膜时向下移动撕裂。这将确保 RPE 将基本保持完整，并且您将获得更干净的撕裂。一旦角膜完全分离，您应该能够看到虹膜和晶状体。用镊子去除虹膜和晶状体，以保持隔离的纯度。要从 RPE 层中去除神经视网膜，请用一把镊子抓住眼罩的外层，并将第二把镊子的手臂放在 RPE 和神经层之间。从那里，轻轻地将神经视网膜从RPE层拉开或舀开，然后丢弃神经视网膜。将 RPE 层移动到培养皿上没有碎屑的其他位置。要将 RPE 与脉络膜和巩膜分离，请用 5/45 式镊子轻轻刮擦眼罩内部，以确保 RPE 细胞的分离。RPE细胞在悬浮和完全RPE细胞生长培养基时呈现混浊。使用3.2毫升无菌转移移液管吸出细胞，并转移到含有完整RPE细胞生长培养基的新15毫升离心管中。细胞可以在室温下以300g离心10分钟。然后，您可以吸出上清液并将沉淀重悬于加热的RPE细胞生长培养基中。将细胞接种在 100 毫米培养皿上，并在 37 摄氏度和 5% CO 2 下孵育。这些图像显示了第0代和第1代的成功分离，这通过RPE细胞的色素沉着很明显。在第4代，细胞的特征性降低，色素减少，表现出纺锤状外观或变得更坚固，表面积更大。我们用RPE特异性标记物RPE65和细胞骨架蛋白F-肌动蛋白验证了RPE细胞。我们还通过用穆勒细胞特异性标记物vimentin染色来检查穆勒细胞的潜在污染。该协议是对现有协议的简化改编。该协议使用大多数研究实验室中可用的材料和相关设备，这将有助于以有效的方式分离原代小鼠RPE细胞。

Research

JoVE Journal

Biology

Isolation of Retinal Stem Cells from the Mouse Eye

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

科研

生物学

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Isolation of Immune Cells from Primary Tumors

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

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

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, ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

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 ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

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 ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

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. ...