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。我们要感谢 Sylvia B. Smith 博士，因为该方案是基于她的 Müller 细胞分离方案的修改版本。

所有动物实验均符合 ARVO 关于在眼科和视觉研究中使用动物的声明，并按照动物护理和使用研究所委员会 （IACUC） 和奥克兰大学政策（协议编号 2022-1160）批准的动物协议进行
1. 培养基和溶液制备
<ol><li>通过在 Dulbecco 改良 Eagle 培养基（DMEM，材料表中的详细信息）中补充 1:1000 稀释的青霉素/链霉素来制备 Müller 细胞眼溶液。例如，对于 10 mL 的 DMEM，添加 10 μL 青霉素/链霉素。 注意:这是一种制备的简单无血清培养基。此外，在用于细胞培养工作之前，将完全培养基在 37 °C 水浴中加热。</li>
	<li>通过在 DMEM 中补充 10% 胎牛血清（FBS，热灭活）和 1% 青霉素/链霉素，制备完整的原代 Müller 细胞生长培养基。例如（500 mL DMEM 培养基 + 50 mL FBS + 5 mL 青霉素/链霉素）。</li>
	<li>用浓度为 200 U/mL 的胶原酶 IV 制备 0.05% 胰蛋白酶-EDTA 溶液（胰蛋白酶-EDTA 用于溶解计算量的胶原酶 IV 粉末以达到所需浓度）。</li>
</ol>2. 眼球摘除术
<ol><li>按照 IACUC 指南使用 CO2 吸入对小鼠进行道德安乐死。<ol><li>简而言之，将动物置于 CO2 室中，以腔室体积/分钟的 30-70% 填充率引入 100% CO2，以达到 2-3 分钟内快速失去知觉的目标，对小鼠的痛苦最小。</li>
		<li>由于小鼠年龄较小 （~10 天），因此将颈椎脱位作为辅助方法，以确保适当的安乐死。</li>
	</ol></li>
	<li>用 70% 乙醇对工作区域消毒后，将鼠标放在无菌垫上。</li>
	<li>将镊子的臂放在眼睛后部，轻轻地从眼眶区域拉出，然后用 5/45 式镊子对眼眶区域施加压力以引起眼球突出，从而取出眼睛。 注意:解剖前用 70% 乙醇消毒解剖工具，然后用磷酸盐缓冲盐水消毒。</li>
	<li>通过缓慢包裹眼睛，确保视神经仍然连接到眼睛。确保将视神经（视觉识别为白索）拉到眼睛后面。 注意:此步骤不需要在显微镜下进行，可以在灭菌的工作台上进行。</li>
</ol>3. 眼摘除的治疗
<ol><li>用铝箔覆盖 15 mL 离心管，并在试管中加入 10 mL Müller 细胞眼溶液。</li>
	<li>将解剖的眼睛放入含有 Müller 细胞眼溶液的 15 mL 试管中，让它们在室温下放置过夜或约 18 小时。</li>
	<li>18 小时后，倒出眼液，并用 2-3 mL 温热 （37 °C） 磷酸盐缓冲盐水 （PBS） 短暂冲洗眼睛。</li>
	<li>倒出 PBS 并将眼睛移至含有 10 mL 胰蛋白酶溶液的 100 mm 培养皿中。（在步骤 1.3 中准备）。 注:胰蛋白酶用于整个去核眼，作为解离剂，在解剖过程中促进细胞与视网膜层分离。解剖的视网膜有许多细胞，包括 RPE 细胞，这些细胞很粘，最有可能污染 Müller 细胞培养物 14。显微镜检查清楚地表明，分离 5 天后（第一次更换培养基期间），RPE 细胞从板中分离并死亡13。用于 RPE 分离的培养基是 DMEM/F-12，其成分与 DMEM（用于 Müller 细胞分离）不同，含有丙酮酸钠和低葡萄糖。</li>
	<li>将培养皿放入培养箱中，在 37 °C 和 5% CO2 下孵育 1.5 至 2 小时。</li>
	<li>孵育后，将眼睛转移到含有大约 5 mL 完整原代 Müller 细胞生长培养基的 60 mm 培养皿中。</li>
</ol>4. 解剖
注:此程序必须在培养罩的无菌环境中进行。因此，必须用 70% 的酒精对通风橱表面以及所有工具和解剖显微镜进行彻底消毒。值得注意的是，该视频是在引擎盖外录制的，以便更好地看到和更清晰的演示目的。
<ol><li>在培养皿中放入一小块消毒的实验室级组织，以帮助在解剖过程中稳定眼睛。将眼睛放在解剖显微镜下开始解剖。 注:使用实验室级组织有助于在充满液体的培养皿（完全培养基）中操纵眼睛，确保精确解剖。</li>
	<li>使用 Vannas 剪刀和 M5S 式镊子去除结缔组织、眼外肌和视神经。<ol><li>用 M5S 风格的镊子抓住眼睛，然后用 Vannas 剪刀或注射器针头在 ora 锯齿切片上切开。 注意:眼睛的前部和后部之间有一条浅蓝色的圆线，称为 ora serrata，可以用作解剖过程中准确方向的标志。</li>
		<li>用 M5S 式镊子抓住切口，从眼睛后部拉出或撕裂角膜。以小增量拉动以确保视网膜的完整性保持完整。 注意:这些步骤是从眼罩的后部（神经和色素视网膜）中去除眼睛的前部（角膜、虹膜和晶状体）。</li>
		<li>为了保持视网膜层的完整性，轻轻拉动并从神经视网膜上去除视网膜色素上皮。神经视网膜应没有任何黑色斑点，以尽可能确保分离的纯度，因为 RPE 层通常是粘性的并附着在神经元视网膜上。</li>
		<li>将解剖的神经视网膜切成小块，然后再将其收集到板中，以确保细胞在板上均匀分布。</li>
	</ol></li>
	<li>将神经视网膜接种在含有 4 mL 完整原代 Müller 细胞生长培养基的 T25 培养瓶中。</li>
	<li>最后，将培养瓶放入培养箱中，并在 37 °C 和 5% CO2 下孵育。</li>
</ol>5. 原代 Müller 神经胶质细胞的培养
<ol><li>3-5 天内不要移动培养瓶，以促进细胞生长。</li>
	<li>第五天后更换培养基，此后每隔一天更换一次，直到细胞达到 90% 汇合。</li>
</ol>6. 原代 Müller 神经胶质细胞的传代
<ol><li>吸出 Müller 细胞培养基，然后在 PBS 中洗涤 2 mL，然后吸出 PBS。加入 2 mL 0.25% 胰蛋白酶-EDTA （1x） 或足以覆盖板。</li>
	<li>在室温下孵育 5-10 分钟。</li>
	<li>确保在显微镜下将细胞从板上分离。然后，收集细胞悬液，并向收集管中加入 4 mL 预热 （37 °C） 完全原代 Müller 细胞生长培养基（在步骤 1.2 中制备）。</li>
	<li>以 300 x g 离心 7 分钟。吸出上清液后，将沉淀重悬于 10 mL 完整的原代 Müller 细胞生长培养基中。因为细胞数量可以根据解剖的眼睛数量而变化，因此，最好在接种和孵育之前对细胞进行计数。推荐的接种密度为 3.0 x 106 个细胞，溶于 T75 培养瓶中。 注:细胞最多可传代 4-5 次。然而，最一致的实验结果是在 1-2 代后发现的。</li>
</ol>7. 免疫荧光
注:使用免疫荧光方案对 Müller 细胞特异性进行染色和验证。以下是免疫荧光实验步骤的简要概述。此步骤在第一次传代12 后执行。
<ol><li>将细胞接种到细胞培养室载玻片中（8 孔室载玻片每孔 30,000 个细胞）。</li>
	<li>将分离的细胞在完整的Müller细胞培养基（在步骤1.2中制备）中于37°C和5%CO2下培养24-48小时。</li>
	<li>当细胞汇合 80-90% 时，吸出培养基并在每个孔上用 250-300 μL 1x PBS 洗涤腔室载玻片。</li>
	<li>在每个孔中用 250-300 μL 4% 多聚甲醛固定载玻片 10 至 15 分钟，然后用 PBS/TX-100 洗涤（每个 5 分钟/3 次）。</li>
	<li>在 37 °C 下加入封闭缓冲液 1 小时（参见 材料表）。</li>
	<li>吸出封闭溶液，然后添加对 Müller 细胞具有特异性的一抗，以使用谷氨酰胺合成酶和波形蛋白 5,12 确认分离细胞的纯度。在 37 °C 下孵育 3 小时（使用 1:100-1:200 的抗体浓度在封闭缓冲液中，体积为 150-200 μL/玻片）。用 PBS/TX-100 洗涤剂洗涤载玻片（各 5 分钟和 10 分钟）。 注:选择这些抗体是因为它们是识别 Müller 细胞和确保 Müller 细胞培养成功和纯度的标志。</li>
	<li>加入二抗（抗体浓度范围为 1:1000，溶于 150-200 μL/玻片的封闭缓冲液中）并在 37 °C 下孵育 1 小时。 然后，用 PBS/Triton X-100 洗涤 3 次（每次 5-10 分钟）。</li>
	<li>滴一滴 DAPI 封固剂以标记细胞核，然后用盖玻片覆盖载玻片。放置盖玻片时避免气泡。</li>
	<li>使用荧光显微镜检查载玻片并捕获图像（参见 材料表）。 注:用 ex/em 359/461 处的 DAPI 以 10 倍放大倍率定位细胞以定位细胞;更高的放大倍数可以捕获细胞。其他波长将取决于所选颜色。绿色 （GFP） - ex/em 488/510。红色 （Cy3）- ex/em 555/569。</li>
</ol>

<ol>
	<li>Liu, Y., Wang, C., Su, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+signaling+in+m&#252;ller+glia:+Progenitor+cells+for+regenerative+and+neuroprotective+responses+in+pharmacological+models+of+retinal+degeneration.">Cellular signaling in m&#252;ller glia: Progenitor cells for regenerative and neuroprotective responses in pharmacological models of retinal degeneration.</a> J Ophth. 2019, 5743109 (2019).</li><li>Goldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glial+cell+reprogramming+and+retina+regeneration.">M&#252;ller glial cell reprogramming and retina regeneration.</a> Nat Rev Neurosci. 15 (7), 431-442 (2014).</li><li>Hamon, A., Roger, J. E., Yang, X. -. J., Perron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glial+cell-dependent+regeneration+of+the+neural+retina:+An+overview+across+vertebrate+model+systems.">M&#252;ller glial cell-dependent regeneration of the neural retina: An overview across vertebrate model systems.</a> Dev Dyn. 245 (7), 727-738 (2016).</li><li>Kobat, S. G., Turgut, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+M&#252;ller+cells.">Importance of M&#252;ller cells.</a> Beyoglu Eye J. 5 (2), 59-63 (2020).</li><li>Liu, X., Tang, L., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+M&#252;ller+cell+isolation+and+culture.">Mouse M&#252;ller cell isolation and culture.</a> Bio Protoc. 7 (15), e2429 (2017).</li><li>Bringmann, 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=M&#252;ller+cells+in+the+healthy+and+diseased+retina.">M&#252;ller cells in the healthy and diseased retina.</a> Prog Ret Eye Res. 25 (4), 397-424 (2006).</li><li>de Melo Reis, R. A., Ventura, A. L. M., Schitine, C. S., de Mello, M. C. F., de Mello, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glia+as+an+active+compartment+modulating+nervous+activity+in+the+vertebrate+retina:+neurotransmitters+and+trophic+factors.">M&#252;ller glia as an active compartment modulating nervous activity in the vertebrate retina: neurotransmitters and trophic factors.</a> Neurochem Res. 33 (8), 1466-1474 (2008).</li><li>Fischer, A. J., Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glia+are+a+potential+source+of+neural+regeneration+in+the+postnatal+chicken+retina.">M&#252;ller glia are a potential source of neural regeneration in the postnatal chicken retina.</a> Nat Neurosci. 4 (3), 247-252 (2001).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+rod+and+cone+cell+death+and+regeneration+in+the+adult+albino+zebrafish+(Danio+rerio)+retina.">Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.</a> J of Neurobiol. 44 (3), 289-307 (2000).</li><li>Shang, P., Stepicheva, N. A., Hose, S., Zigler, 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> J of Vis Exp. (133), 56997 (2018).</li><li>Sarthy, P. V., Bunt, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultrastructure+of+isolated+glial+(M&#252;ller)+cells+from+the+turtle+retina.">The ultrastructure of isolated glial (M&#252;ller) cells from the turtle retina.</a> Anat Rec. 202 (2), 275-283 (1982).</li><li>Shanmugam, A. K., 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=Progesterone+receptor+membrane+component+1+(PGRMC1)+expression+in+murine+retina.">Progesterone receptor membrane component 1 (PGRMC1) expression in murine retina.</a> Curr Eye Res. 41 (8), 1105-1112 (2016).</li><li>Moustafa, 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=12-HETE+activates+M&#252;ller+glial+cells:+The+potential+role+of+GPR31+and+miR-29.">12-HETE activates M&#252;ller glial cells: The potential role of GPR31 and miR-29.</a> Prostag Oth Lipid M. 171, 106805 (2024).</li><li>Pereiro, X., Ruzafa, N., Acera, A., Urcola, A., Vecino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+method+to+isolate+and+culture+adult+porcine,+rats+and+mice+m&#252;ller+glia+in+order+to+study+retinal+diseases.">Optimization of a method to isolate and culture adult porcine, rats and mice m&#252;ller glia in order to study retinal diseases.</a> Front Cell Neurosci. 14, 7 (2020).</li></ol>

作者没有与本文内容相关的利益冲突声明。

我们实验室先前记录了从小鼠中分离出原代视网膜色素上皮 （RPE）。本手稿描述了原代 Müller 细胞分离的详细演示方案。该程序包括从小鼠眼睛中分离的 Müller 细胞的摘除、处理、解剖、收集、接种、培养和表征。它基于先前在早期出版物中发现的成功方案以及我们在最近出版物中使用的修改后的方案1， 2,13.虽然步骤很简单，但成功的 Müller 细胞分离需要遵守特定的限制和基本要求。经过多次尝试和错误尝试优化该方案，确定了影响其成功的多个因素。
正如我们在实验中观察到的那样，理想的小鼠年龄在 5-11 天之间，以便将分离的细胞生长到四代。培养还受到分离和培养的眼睛数量的影响。对于单次分离，至少需要两只眼睛才能获得足够数量的细胞才能生长和繁殖。另一个重要方面是眼球摘除和接种之间的持续时间;因此，此步骤需要相对较快地执行。据观察，长时间操作眼组织对细胞粘附在培养瓶上和繁殖的能力有不利影响。为了确保分离细胞的距离足够接近以促进增殖，我们建议使用 T25 培养瓶。完成第一次传代后，即可将细胞接种在 T75 上。此外，分离和接种后，从培养箱中取出组织培养瓶或更换培养基至少 5 天，不应干扰细胞。最后，细胞在停止粘附之前只能通过大约四次。虽然这个过程简单易行，但它有一些限制，包括小鼠年龄、细胞传代次数有限等。
Müller 细胞分离的方案并不多，但该方案与可用的方案存在一些显着差异 5,14。首先，摘除和分离之间的时间明显不同。其他方案建议在眼球摘除后尽快进行解剖，通常在 10 分钟内。而目前的方案建议至少 18 小时的静置期，因为这为该方案中的隔离提供了最佳结果。该方案与其他方案之间的另一个显着区别是用酶处理眼睛的时间点。在该方案中，在进行任何解剖之前，甚至在去除结缔组织和眼外肌之前，对眼睛进行治疗。而其他方案在酶处理之前将视网膜与眼睛分离。此外，酶配方也不同。在该方案中，酶溶液由胰蛋白酶和 IV 型胶原酶组成。在类似的方案中，木瓜蛋白酶和 DNase 用于组织消化。此外，培养容器不同，该方案建议在第一次传代后在 T25 上培养，然后在 T75 上培养。而其他方案使用培养皿或多孔板。最后，该方案建议不应干扰培养瓶（5-7 天）以促进细胞生长。而在其他协议中，介质更换是在一天后执行的。

Müller 细胞 （MCs） 是在视网膜组织中发现的主要和最丰富的神经胶质细胞。它们是在视网膜内提供结构完整性和代谢功能的关键参与者1。MC 的战略结构分布在整个视网膜厚度上，从而为视网膜提供支持。除了支架状特性外，它们还具有视网膜神经元的代谢功能，为它们提供能量底物，包括葡萄糖和乳酸。这些功能对于维持健康的神经元功能至关重要。据报道，受损的 MC 会导致各种视网膜疾病，包括年龄相关性黄斑变性、糖尿病性视网膜病变和青光眼 2,3。MC 可以是视网膜再生治疗的内源性细胞来源1。它们也构成了视网膜的很大一部分，强有力的证据表明，在几个物种中，这些细胞可以被刺激以替代缺失的神经元2。它们与神经元有益地合作，圆锥形分支的 Müller 细胞末端密集地展开血管并连接视网膜的神经成分。为了维持神经元发育和神经元可塑性，Müller 细胞充当神经元的软基质，保护它们免受机械损伤3。此外，在病理情况下，Müller 细胞可能会分化为神经祖细胞或干细胞，复制或再生丢失的光感受器和神经元 2,3,4。Müller 细胞保留了视网膜干细胞的特性，包括不同程度的自我更新和分化潜力 5,6。Müller 神经胶质细胞具有重要的视网膜谱系，可产生神经营养因子、摄取和回收神经递质、在空间上缓冲离子并维持血视网膜屏障，以保持视网膜处于体内平衡 7,8,9。这凸显了 Müller 细胞作为治疗视网膜变性相关疾病的细胞疗法中一种有前途的工具的潜力。Müller 细胞是分布在整个视网膜中的原代神经胶质细胞，与神经元和血管相连。它们起着至关重要的保护作用，提供必要的结构和代谢支持，以维持视网膜细胞的活力和稳定性。遗憾的是，在文献中发现的从视网膜中分离原代 Müller 细胞的方案很少 10,11。
我们提出了一种可靠的分离和培养小鼠原代 Müller 细胞的增强方法。我们小组使用该方案从野生型 C57BL/6 小鼠和转基因小鼠中分离 Müller 细胞12,13。年龄在 5 至 11 天之间，无性别偏好的小鼠用于该方案。细胞已传代多达 4 次;然而，在 P4 时，它们不再粘附在培养瓶上，因此很难培养出健康的培养物。培养物通常被视网膜色素上皮 （RPE） 细胞污染，因此在对细胞系进行任何其他实验之前，细胞应至少传代一次。传代允许进一步分离污染物。因此，提出的方案提供了一种快速有效的方法来分离小鼠 Müller 细胞，然后可以将其用作研究治疗靶点和评估视网膜疾病潜在治疗方法的可靠平台14。

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	<tbody>
		<tr>
			<td>Beaker : 100mL</td>
			<td>KIMAX</td>
			<td>14000</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV&#160;</td>
			<td>Worthington&#160;</td>
			<td>LS004188</td>
			<td></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 (1X)&#160;</td>
			<td>Thermo Scientific</td>
			<td>11885084</td>
			<td>Media to grow M&#252;ller cells&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>gibco</td>
			<td>26140079</td>
			<td>For complete Muller cell culture media</td>
		</tr>
		<tr>
			<td>Glutamine synthase</td>
			<td>Cell signalling</td>
			<td>80636</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VISO 160i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></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>Pen Strep</td>
			<td>gibco</td>
			<td>15140-122</td>
			<td>For complete M&#252;ller cell culture media</td>
		</tr>
		<tr>
			<td>Phosphate Buffer saline (PBS)</td>
			<td>Thermo Scientific</td>
			<td>J62851.AP</td>
			<td></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>
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		<tr>
			<td>Suspension Culture Dish 60mm x 15mm Style</td>
			<td>Corning</td>
			<td>430589</td>
			<td></td>
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		<tr>
			<td>Tissue Culture Dish : 100x20mm style</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
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		<tr>
			<td>Tornado Tubes: 15mL</td>
			<td>Midsci</td>
			<td>C15B</td>
			<td></td>
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		<tr>
			<td>Tornado Tubes: 50mL</td>
			<td>Midsci</td>
			<td>C50R</td>
			<td></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>
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		<tr>
			<td>Underpads, Moderate : 23&#34; X 36&#34;</td>
			<td>McKesson</td>
			<td>4033</td>
			<td></td>
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		<tr>
			<td>Vannas Spring Scissors - 2.5mm Cutting Edge</td>
			<td>FST</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin&#160;</td>
			<td>invitrogen</td>
			<td>MA5-11883</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss AxioImager Z2</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
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		<tr>
			<td>Zeiss Zen Blue 2.6</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
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</table>

isolation of primary mouse retinal glial m&#252;ller cells

视网膜的主要支持细胞是视网膜神经胶质 Müller 细胞。它们覆盖整个视网膜表面，并且靠近视网膜血管和视网膜神经元。由于它们的生长，Müller 细胞在健康的视网膜中执行几项关键任务，包括神经递质、视黄酸化合物和离子（如钾 K+）的摄取和回收。除了调节血液流动和维持血视网膜屏障外，它们还调节新陈代谢和视网膜的营养供应。本手稿介绍了分离原代小鼠 Müller 细胞的既定程序。为了更好地了解各种眼部疾病小鼠模型所涉及的潜在分子过程，Müller 细胞分离是一种很好的方法。本手稿概述了从小鼠中分离 Müller 细胞的详细程序。从去核到播种，整个过程持续大约几个小时。接种后 5-7 天内，不应更换培养基，以使分离的细胞不受阻碍地生长。接下来使用形态学和不同的免疫荧光标记物进行细胞表征。细胞的最大传代次数为 3-4 次。

验证分离的 Müller 细胞的特异性、纯度和屏障功能 为了确认分离的 Müller 细胞的活力、形态和独特品质，在光学显微镜下检查了这些细胞。记录 P0 和 P1 图像（图 1A）。为了检查分离的 Müller 细胞是否受到 RPE 细胞的污染并确认其纯度，使用对 RPE 细胞标志物 RPE65 具有特异性的抗体进行免疫荧光染色 （IF）。人视网膜色素细胞 （ARPE -19） 用作阳性对照。RPE65 染色的 RPE 细胞（红色和蓝色用于核染色）如图 1B 所示（下图）。RPE65 特异性抗体未对分离的 Müller 细胞进行染色（图 1B，上图）。RPE65 对 ARPE-19 细胞（红色）进行染色而没有对分离的细胞进行染色，这一事实表明分离的细胞不是 RPE 细胞。培养基中丙酮酸和低葡萄糖的存在为 RPE 细胞的生长创造了不利条件。因此，即使在 RPE 细胞污染的情况下，它们最终也会从培养瓶中分离。
此外，为了确认分离细胞的身份，使用 Müller 细胞特异性标志物的 Müller 细胞免疫荧光染色来确认 Müller 细胞的成功分离。使用了称为波形蛋白的细胞骨架中间丝蛋白12,13。此外，还使用了谷氨酰胺合成酶 （GS），它催化谷氨酸和氨的缩合反应形成谷氨酰胺，作为视网膜神经胶质 Müller 细胞的关键功能5。
总的来说，分离的细胞对 RPE65 染色阴性（红色，图 1B），波形蛋白阳性（绿色，图 1C）和 GS（绿色，图 1D）。IF 染色证实了分离的 Müller 细胞的成功分离（纯度和特异性）。图 1E 是波形蛋白（上图）和 GS（下图）的阴性对照，证实了抗体的特异性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66237/66237fig01.jpg"> 图 1:Müller 细胞分离的验证（纯度和特异性）。 （A） 传代的光学显微镜图像:传代零 （P0） 和 （P1） 形态。（B） RPE65 免疫染色联合 DAPI 核染色。（C） 高倍率 DAPI 核染色的波形蛋白免疫染色。（D） 相同高放大倍率下的 GS 免疫染色。（E） 阴性对照，以确认波形蛋白和 GS 抗体染色的特异性。比例尺 = 分别为 300 μm、300 μm、50 μm、20 μm、20 μm、50 μm、50 μm。 <a href="https://www.jove.com/files/ftp_upload/66237/66237fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

本手稿描述了从小鼠眼睛中分离视网膜神经胶质 Müller 细胞的详细方案。该方案从小鼠眼睛的摘除和解剖开始，然后是 Müller 细胞的分离、接种和培养。

原代小鼠视网膜神经胶质 Müller 细胞的分离

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both ...

isolation-of-primary-mouse-retinal-glial-mller-cells

Nanyang Technological University

Research

JoVE Journal

Biology

Isolation of Primary Mouse Retinal Glial M&#252;ller Cells

1.1K 次观看.  Oakland University. 本手稿描述了从小鼠眼睛中分离视网膜神经胶质 Müller 细胞的详细方案。该方案从小鼠眼睛的摘除和解剖开始，然后是 Müller 细胞的分离、接种和培养。

视频: 原代小鼠视网膜神经胶质 Müller 细胞的分离

Isolation of Retinal Stem Cells from the Mouse Eye

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 non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

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

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 in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel 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 and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

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

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 week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic 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 ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

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

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 ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

成年小鼠黏膜下层和固有层肠道胶质细胞的分离

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

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 ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

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. ...