Name: optimizing mouse primary lens epithelial cell culture: a comprehensive guide to trypsinization
Uploaded: 2024-06-21T12:30:00.000+00:00
Duration: 3 min 47 s
Description: Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, ...

这项工作得到了NEI R21EY033941（对吴洪利）的支持;国防部W81XWH2010896（致吴洪利）;R15GM123463-02 （致Kayla Green和Hongli Wu）

所有动物实验均按照视觉和眼科研究协会关于在眼科和视觉研究中使用动物的指南进行。程序批准由北德克萨斯大学健康科学中心动物护理和使用委员会（协议编号：IACUC-2022-0008）授予。这些研究使用了通常低于2周龄的年轻C57BL / 6J小鼠。
1.培养基制备及晶状体解剖
<ol><li>通过向 450 mL DMEM 中加入 50 mL 胎牛血清 （FBS） 和 0.1 mL 50 mg/mL 庆大霉素来制备培养基。</li>
	<li>人道地对小于2周的C57BL / 6J小鼠实施安乐死。 注意：我们使用 CO2 吸入法实施安乐死。CO2 安乐死系统的最佳流速应位移 30% 至 70% 的腔室或笼容积/分钟。</li>
	<li>用手术剪刀轻轻取下眼睑，并用弯曲的镊子在眼窝的两侧轻轻按压，使眼睛向外突出。使用白内障刀在角膜上仔细切开，然后用弯曲的镊子小心地取出晶状体，确保不会损坏晶状体或其囊。 注意： 执行这些步骤时要小心，以保持镜头囊的完整性。由于镜片的脆弱性，使用带有弯曲和钝头的解剖工具非常重要，以最大限度地降低镜片损坏的风险。</li>
	<li>使用带有钝头的弯曲镊子将镜片转移到 60 mm 塑料组织培养皿中，该培养皿中装满了 5 mL 预热和无菌的 Dulbecco 磷酸盐缓冲盐水 （DPBS） 溶液，其中含有 10 μg/mL 庆大霉素。</li>
	<li>用含有 10 μg/mL 庆大霉素的 DPBS 溶液轻轻冲洗镜片，以去除任何潜在的碎屑或污染物，为进一步处理镜片做好准备，并保持无菌培养环境。</li>
	<li>为了获得足够数量的 LEC，将 4 个透镜用于 24 孔培养板，将 6 个透镜用于 6 孔培养板。</li>
</ol>2. LECs隔离
<ol><li>完成漂洗过程后，将镜头放在一张滤纸上，让它干燥。</li>
	<li>镜片充分干燥后，小心地将其转移到培养皿的盖子上，为取出镜片胶囊做准备。</li>
	<li>向上旋转晶状体，确保前节朝上。在使用镊子夹住前囊的同时，使用惯用手的囊镊子在囊中产生一个小撕裂。向相反方向轻轻拉动两个工具以取出胶囊并将其放入 DPBS 中，直到完成所有晶状体解剖。 注意：为避免任何差异，研究人员应立即解剖每个晶状体上皮囊并将它们暂时储存在 DPBS 中。只有在完成所有解剖后，胶囊才能集体转移到保持在37°C的胰蛋白酶中，确保同步和均匀的暴露。</li>
	<li>小心地将镜头囊转移到 6 孔板上。向每个孔中加入 1 mL 0.05% 胰蛋白酶溶液以启动酶解过程。</li>
	<li>轻轻搅拌胰蛋白酶溶液以确保均匀渗透。将板置于细胞培养箱中，让胶囊在37°C下消化8-10分钟。 注意：此步骤有助于晶状体囊组织的分解和随后单个上皮细胞的释放。</li>
	<li>孵育后，使用解剖剪刀小心地切碎消化的晶状体胶囊，以分解任何残留的组织团块并促进细胞分离。 注意：强调组织切碎的彻底性，以确保从消化的晶状体胶囊中有效释放细胞。</li>
	<li>加入 0.5 mL 含有 10% FBS 的培养基以淬灭胰蛋白酶。将组织样品转移到离心管中，并以1,000× g 离心5分钟。</li>
	<li>小心地除去上清液，不要干扰细胞沉淀。使用 1 mL 培养基重悬细胞并将细胞接种在 24 孔板中。</li>
	<li>每 2-3 天更换一次培养基。</li>
</ol>3. LECs亚文化
<ol><li>一旦细胞达到汇合，从培养皿中取出培养基。继续用 1 mL DPBS 洗涤细胞 2 次。</li>
	<li>加入 200 μL 胰蛋白酶-EDTA 溶液，将细胞置于培养箱中 5 分钟。</li>
	<li>孵育后，将细胞从培养箱中取出并在显微镜下检查它们，以确认它们已从培养皿中分离并开始漂浮。</li>
	<li>加入 1 mL 培养基，轻轻移液细胞 3-5 倍以分离所有细胞。</li>
	<li>将细胞转移到离心管中，并以1,000× g 离心5分钟。</li>
	<li>小心地除去上清液并将细胞重悬于完整的生长培养基中。</li>
	<li>如果需要，使用血细胞计数器计数细胞数量。</li>
	<li>以 1：2 或 1：3 的比例细分细胞悬液以进行传代培养。</li>
	<li>当培养物再次汇合时，重复上述过程。 注意：LEC在高密度培养条件下茁壮成长。避免过度稀释细胞，因为这可能会阻碍它们的生长。</li>
</ol>4.储存和运输
注意：理想的存储单元数是 ~1 × 106。
<ol><li>用 1 mL DPBS 彻底洗涤细胞 3 次。洗涤后，加入 1 mL 胰蛋白酶-EDTA 溶液，并将细胞置于培养箱中 5 分钟。</li>
	<li>加入 2 mL 完全培养基，将细胞悬液转移到离心管中，并以 1,000 × g 离心 5 分钟。</li>
	<li>弃去上清液并将细胞重悬于由 90% FBS 和 10% DMSO 组成的冷冻培养基中，目标是细胞密度为 1 ×10 6 个细胞/mL。将细胞悬液转移到冷冻管中。</li>
	<li>立即将细胞移至-20°C环境中1小时，然后在-80°C下过夜，然后永久储存在液氮中。 注意：如果没有液氮，则可以在-80°C下初始一小时后将细胞储存在-20°C。</li>
	<li>如果需要发货，请将冷冻管中的细胞装在装有干冰的包装中，以便隔夜交付。</li>
	<li>收到细胞后，确保快速回收并将细胞置于传代培养中。如果立即培养不可行，则将细胞转移到液氮中进行长时间储存。 注意： 如果要运送细胞，请确保样品深埋在干冰中，以防止温度波动造成潜在损坏。</li>
</ol>5. LEC验证
<ol><li>将LEC板入带有盖玻璃的35mm培养皿中，并培养约48小时。</li>
	<li>用PBS洗涤细胞2次，并在-20°C下用冷甲醇固定细胞10分钟。</li>
	<li>用PBS洗涤固定细胞3×5分钟，并在室温下用封闭缓冲液孵育固定细胞1小时，以防止非特异性结合。</li>
	<li>封闭后，将细胞在4°C下与一抗（αA-晶状体蛋白，γ-晶状体蛋白和PROX1抗体）在稀释缓冲液中以1：50的比例单独稀释。</li>
	<li>用PBS洗涤细胞3×5分钟，并将二抗在稀释缓冲液中以1：100稀释的二抗孵育1小时。</li>
	<li>用PBS洗涤细胞3×5分钟，并在室温下用PBS中的5μg/ mLHoechst 33342染色细胞10分钟以观察细胞核。</li>
	<li>用PBS洗涤细胞2×5分钟以除去多余的染色溶液，并使用荧光显微镜捕获细胞的荧光图像，使用DAPI通道用于细胞核，FITC通道用于αA-，γ-晶状体蛋白和PROX1。</li>
</ol>

晶状体上皮细胞的培养和表征

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本文介绍的方案为成功分离、培养和传代 LEC 提供了全面的分步指南，并附有视频文档。详细的视觉指南和书面说明增强了协议的清晰度和可访问性，促进了该领域研究人员的使用和可重复性。最终目的是促进围绕 LEC 在白内障形成和 PCO（白内障手术后普遍存在的并发症）中的作用的知识体系的扩展。
当将原代 LEC 与晶状体上皮细胞系（如 HLE-B3 和 SRA01/04）进行比较时，每种?...

眼睛的晶状体通过将入射光聚焦到视网膜上，在视觉中起着至关重要的作用。它由透明的无血管结构组成，由特化细胞组成，其中晶状体上皮细胞 （LEC） 是关键参与者。LEC 位于晶状体的前表面，负责保持其透明度、调节水平衡并参与晶状体生长和发育 1,2。LEC 是位于晶状体前部的一种独特类型的细胞，通过在整个生命周期中持续产生晶状体纤维，在维持晶状体清晰度和功能方面发挥着关键作用。
白内障的特征是晶状体逐渐混浊，导致光线失真和散射，导致视力受损 3,4。白内障形成的确切机制是复杂和多因素的，涉及各种细胞和分子过程，如紫外线辐射、氧化损伤和糖基化 5,6。已发现 LEC 对白内障的发展有显着贡献，使其成为研究的重要焦点 1,2,7,8,9。
此外，当今眼科最紧迫的问题之一是后囊混浊 （PCO） 的发生率相对较高，也称为继发性白内障。PCO 仍然是白内障手术后最常见的并发症，在手术后 5 年内影响多达 20-40% 的成人患者和 100% 的儿童10.PCO主要是由白内障摘除术后残留在囊袋中的LEC引起的。这些细胞经历多方面的病理生理学转化，不仅涉及上皮-间充质转化 （EMT），还涉及 LEC 向晶状体纤维的分化，从而形成 LEC、纤维和肌成纤维细胞的混合物细胞群 11,12,13。转化的细胞增殖并迁移到晶状体后囊，导致视力障碍。了解培养模型中 LEC 的行为和控制机制可以为 PCO 的预防和管理提供有价值的见解。因此，这种培养 LEC 的方案为眼科研究人员提供了一个重要的工具，旨在研究、理解并最终对抗这种普遍的术后并发症。
为了揭示LEC生物学的复杂性及其在白内障形成和PCO中的作用，必须建立强大且可重复的 体外 原代细胞培养系统。原代 LEC 培养为研究人员提供了一个受控环境来研究 LEC 的功能、信号传导和分子特征。此外，它还允许研究细胞过程和不同实验条件的影响，为晶状体生理学和病理学提供有价值的见解。
先前的研究丰富了我们对LEC培养技术的理解14,15,16,17,18,19,20。尽管这些研究采用了各种方法，并在LEC行为和特征方面取得了重要发现，但目前的文献中缺乏用于培养LEC的全面且可访问的视频记录方案。这种局限性会阻碍新手研究人员准确重现技术的能力，并可能导致实验结果的不一致和变化。通过提供视频录制协议，本文旨在弥合这一差距，并提供一种标准化资源，以提高LEC文化领域的可重复性并促进知识转移。

<table><tbody><tr><td>0.05% Trypsin-EDTA</td><td>Thermo Fisher</td><td>#25300054</td><td>For LECs dissociation</td></tr><tr><td>Alexa Fluor 488 Secondary Antibody&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>#715-545-150</td><td>For cell validation</td></tr><tr><td>Alexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>111-605-003</td><td>For cell validation</td></tr><tr><td>Antibody dilution buffer</td><td>Licor</td><td>#927-60001</td><td>For cell validation</td></tr><tr><td>Beaver safety knife</td><td>Beaver-Visitec International</td><td>#3782235</td><td>For lens dissection</td></tr><tr><td>Blocking buffer</td><td>Licor</td><td>#927-60001</td><td>For cell validation</td></tr><tr><td>Capsulorhexis forceps</td><td>Titan Medical Instruments</td><td>TMF-124</td><td>For lens capsule isolation</td></tr><tr><td>DMEM</td><td>Sigma Aldrich</td><td>D6429</td><td>For LECs culture medium</td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>#D2650</td><td>For making freezing medium&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline&amp;nbsp;</td><td>Thermo Fisher</td><td>#J67802</td><td>For lens dissection</td></tr><tr><td>Dumont tweezers</td><td>Roboz Surgical Instrument</td><td>RS-4976</td><td>For lens capsule isolation</td></tr><tr><td>EpiCGS-a (optional)</td><td>ScienCell</td><td>4182</td><td>For LECs culture medium</td></tr><tr><td>FBS</td><td>Sigma Aldrich</td><td>F2442</td><td>For LECs culture medium</td></tr><tr><td>Gentamicin (50&amp;nbsp;mg/mL)</td><td>Sigma-Aldrich</td><td>G1397</td><td>For LECs culture medium</td></tr><tr><td>Hoechst 33342 solution</td><td>Thermo Fisher</td><td>#62249</td><td>For cell validation</td></tr><tr><td>Micro-dissecting scissors</td><td>Roboz Surgical Instrument&amp;nbsp;</td><td>RS-5983</td><td>For lens dissection</td></tr><tr><td>Micro-dissecting tweezers</td><td>Roboz Surgical Instrument&amp;nbsp;</td><td>RS5137&amp;nbsp;</td><td>For lens dissection</td></tr><tr><td>PROX1 antibody</td><td>Thermo Fisher</td><td>11067-2-AP</td><td>For cell validation</td></tr><tr><td>Vannas micro-dissecting spring scissors</td><td>Roboz Surgical Instrument</td><td>RS-5608</td><td>For lens capsule isolation</td></tr><tr><td>&amp;alpha;A-crystallin antibody</td><td>Santa Cruz</td><td>sc-28306</td><td>For cell validation&amp;nbsp;</td></tr><tr><td>&amp;gamma;-crystallin antibody</td><td>Santa Cruz</td><td>sc-365256</td><td>For cell validation</td></tr></tbody></table>

optimizing mouse primary lens epithelial cell culture: a comprehensive guide to trypsinization

晶状体上皮细胞（LECs）在维持晶状体的稳态和正常功能方面发挥着多种重要作用。LEC 决定了晶状体的生长、发育、尺寸和透明度。相反，功能失调的 LEC 可导致白内障形成和后囊混浊 （PCO）。因此，建立强大的原代LEC培养系统对于从事晶状体开发、生物化学、白内障治疗和PCO预防的研究人员非常重要。然而，由于原代LECs的可用性有限、增殖速度慢且性质脆弱，其培育长期以来一直面临挑战。 
本研究通过提出原代 LEC 培养的综合方案来解决这些障碍。该方案包含基本步骤，例如配制优化的培养基、晶状体胶囊的精确分离、胰蛋白酶消化技术、传代培养程序、收获方案以及储存和运输指南。在整个培养过程中，使用相差显微镜监测细胞形态。 
为了确认培养的LEC的真实性，进行了免疫荧光测定以检测关键晶状体蛋白（即αA和γ晶状体蛋白）的存在和亚细胞分布。这一详细的方案为研究人员提供了培养和表征原发性 LEC 的宝贵资源，从而促进了我们对晶状体生物学的理解和晶状体相关疾病治疗策略的开发。

如 图2所示，通过遵循该方案，来自C57BL / 6J小鼠的原代LEC在4小时内粘附在培养皿上。值得注意的是，有其他组织的可见残留物，例如后囊和晶状体纤维细胞的部分。然而，这些意想不到的元素并没有附着在培养皿上，因此可以通过改变培养基来去除。随后，在第三天和第五天之间，LEC开始了其扩散阶段。然后，在第七天和第十天之间可以观察到快速增长，这是对数增殖阶段...

作者聚焦：揭开白内障的秘密 – 研究晶状体上皮细胞中的氧化还原修复酶

本手稿概述了用于培养原晶状体上皮细胞 （LEC） 的详细视频方案，旨在提高可重复性并帮助白内障和后囊混浊 （PCO） 的研究。它提供了有关晶状体解剖、LEC 分离和验证的分步说明，可作为有价值的指南，尤其是对于该领域的新手而言。

优化小鼠原晶状体上皮细胞培养：胰蛋白酶消化综合指南

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, ...

我们的研究目标是探索白内障的根本原因，特别强调氧化还原修复酶的功能。我们的目标是确定这种酶在晶状体保护中发挥的关键作用，并确定它们作为白内障预防和治疗药物靶点的潜力。在许多令人兴奋的技术中，我们的研究使用晶状体上皮细胞作为基础平台。这种方法对于揭示白内障形成、后囊混浊和发现新的抗白内障药物背后的机制至关重要。展望未来，我们将专注于了解抗氧化酶和衰老对镜片功能的影响。我们的目标是发现创新的抗氧化疗法，可以延缓晶状体老化，预防白内障，并为PCO提供有效的干预。

optimizing-mouse-primary-lens-epithelial-cell-culture-comprehensive

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization

1.7K 次观看.  University of North Texas Health Science Center. 我们的研究目标是探索白内障的根本原因，特别强调氧化还原修复酶的功能。我们的目标是确定这种酶在晶状体保护中发挥的关键作用，并确定它们作为白内障预防和治疗药物靶点的潜力。在许多令人兴奋的技术中，我们的研究使用晶状体上皮细胞作为基础平台。这种方法对于揭示白内障形成、后囊混浊和发现新的抗白内障药物背后的机制至关重要。展望未来，我们将?...

视频: 作者聚焦：揭开白内障的秘密 – 研究晶状体上皮细胞中的氧化还原修复酶

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

科研

发育生物学

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

医学

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

大规模培育三维重建人类表皮

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

生物工程

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 ...

神经科学

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

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

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

小鼠视网膜色素上皮细胞的原代培养

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

生物学

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

小鼠表皮角质细胞的分离及其体外克隆培养物

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

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 ...

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. ...

优化小鼠原晶状体上皮细胞培养：胰蛋白酶消化综合指南

副本

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此视频中的章节

通过微分试穿素生成马赛克乳腺有机体

移植到小鼠卵巢脂肪垫

大规模培育三维重建人类表皮

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

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

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

小鼠视网膜色素上皮细胞的原代培养

小鼠表皮角质细胞的分离及其体外克隆培养物

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

猪视网膜色素上皮细胞原代培养