我们感谢社论援助乔娜埃利斯。项目描述奖号码R01MH079751（主持人:F.佩鲁齐）从国家心理健康研究所的支持。内容完全是作者的责任，并不代表国家精神卫生研究所或国立卫生研究院的官方意见。

1。聚-D-赖氨酸（PDL）的制备<ol><li>加入5毫升无菌DDH 2至5毫克的客运专线，获得1毫克/毫升的原液。 </li><li>混合原液吹打几次。 </li><li>立即使用或储存聚-D-赖氨酸的解决方案，在2-8°C。 </li></ol> 2。聚-D-赖氨酸（PDL）:涂层塑料细胞培养皿<ol><li> PDL原液稀释用无菌DDH 2 O的终浓度为10微克/毫升。 </li><li>吸取足够的解决方案，覆盖养殖水面面积为60毫米的菜（3毫升）到60毫米的菜。 </li><li>轻轻摇动，以确保甚至文化表面的涂层。 </li><li>涂层板在室温（RT）孵育过夜。 </li><li>第二天，通常一天的清扫，清除吸聚-D-赖氨酸的解决方案，并用3毫升无菌DDH 2 O简要重复此步骤。第二洗净后，完全去除水中的共同愿望。 </li><li>板可以存储长达三个星期在4°C。 </li></ol> 3。聚-D-赖氨酸（PDL）和层粘连蛋白:两玻片制备及玻璃涂料<ol><li>在无菌DDH 2 O的混合客运专线（1毫克/毫升）和层粘连蛋白（1毫克/毫升）储备溶液至终浓度为10和5微克/毫升，分别。 </li><li>吸取足够的成井的玻璃两腔的幻灯片，涵盖文化表面积（1毫升），每2以及双室玻璃幻灯片以及解决方案。 </li><li>轻轻摇动，以确保甚至文化表面的涂层。 </li><li>涂层板，在室温下孵育过夜。 </li><li>第二天，删除聚-D-赖氨酸，层粘连蛋白涂层解决方案，通过吸入和简要洗两次以1毫升无菌DDH 2 O第二洗净后，完全去除水中的共同愿望。 </li><li>玻片可以存储长达三个星期在4°C。 </li></ol>注:任何玻璃室幻灯片，可涂跟随着g此协议。我们经常使用的两玻片，因为每张幻灯片提供的控制测试实验设置（如未经处理与治疗，未转染与转染）。 4。神经解剖和文化<ol><li>在37℃水浴中预热的下列试剂: <ul type="disc"><li>其原来的100毫升瓶装TrypLE快。 </li><li> Neurobasal/B27完全培养基（ 见表一）。成交量回暖取决于电镀（如30毫升十60毫米镀菜）菜肴的数目。 </li></ul></li><li> 4个60毫米培养皿和13毫升至15毫升的BD猎鹰高清晰度聚丙烯锥形管加入3毫升的冷休眠E解决方案。 </li><li>添加25-30毫升冷夹层中（ 见表二 ，个人通信，博士奥林匹亚梅乌奇）三个100毫米培养皿。这些板块，含有大量的中等，将用于冲洗后立即胚胎去除羊膜囊（步骤4.7和4.8）。 </li><li>安乐死的CO 2孕鼠E17的定时根据公共卫生服务政策上的人文关怀和实验动物的使用和在制度上批准的动物护理和使用协议。 </li><li>喷用70％乙醇的小腹和中间穿过一双剪刀暴露子宫和胚胎的皮肤和肌肉。 </li><li>所有胎儿取出，并放置在无菌的100毫米菜含有超过冷夹层中（25-30毫升，见步骤4.3）。 </li><li>削减使用的小剪刀从羊膜囊的胚胎，他们将在第二个100毫米冷夹层介质的菜。 </li><li>在室温下轻轻倾斜5-10秒100毫米的菜洗胚胎。然后，漂洗胚胎转移到第三个100毫米的菜，含夹层介质。清洗多余的介质通常足以消除血液中的所有痕迹。然而，如果NEC需要的话，洗更多的时间，用100毫米的菜新鲜，含25-30毫升冷夹层介质。 </li><li>使用体视显微镜和弯曲钳，每只老鼠胚胎的大脑提取拉出皮肤和颅骨。放置在一个60毫米的菜肴与冷休眠大肠杆菌（通常情况下，与菜不超过5％的大脑）置于冰上这些板块的整个大脑。 </li><li>在一次菜，在解剖显微镜下，半球分开的，孤立的大脑皮质，消除脑和脑膜。 </li><li>可选:沿中线切大脑，提取海马，并按照以下步骤分离海马神经元。 </li><li>收集了15毫升清楚的锥形管含有13毫升冷休眠大肠杆菌的离开大脑皮层上冰，直到完成所有的夹层中的所有解剖皮质。由于其体积小，解剖海马可以在1.5毫升的Eppendorf管，而不是一个15毫升的管收集。如果需要在这一步的皮质，或海马可放置在cryotube含有1毫升的小瓶的Hibernate E + 2％B27的+庆大霉素（50微克/毫升）+ Fungizone 2-4皮质或2-4小瓶海马的比例（250毫微克/毫升）。脑组织，可存放于4℃在黑暗中长达一个星期（稍后的时间还没有被测试）。当需要时，用细钳脑组织转移至15毫升管中的Hibernateé，然后按照下面的协议，分离神经元。 </li><li>管转移到组织文化引擎盖。允许皮质解决管底部，然后小心取出上清液。 </li><li> 15毫升锥形管加入13毫升的新鲜休眠E，允许的皮质定居在试管底部，小心取出上清液。重复此步骤2次以上，最后一次洗涤后，小心地取出所有的媒体。 </li><li>酶消化，加入1-2毫升（根据数量的皮质;使用海马隔离）大脑皮质温暖训练班ypLE快。用封口膜密封管帽和管漂浮在37℃水浴10分钟。 </li><li>喷用70％乙醇管前盖打开，并添加10毫升的Hibernate E.允许皮质定居在试管底部，去除上清。重复此步骤三次洗出TrypLE快。在最后一步，小心地取出所有介质。 </li><li>轻轻磨碎（4-5倍）在2毫升的Neurobasal/B27完全培养基的皮质，用火抛光玻璃巴​​斯德（直径约1毫米）。要小心，以避免气泡。 </li><li>重复使用无菌玻璃巴斯德吸管直径较小（即约1/2-3/4毫米直径的吸管）另外的4-5倍。不要使用巴斯德吸管比这个小，它会破坏细胞。 </li><li>允许组织残片（一般很少，如果有的话）来解决。 </li><li>转移到一个新的15 ml管上的单细胞悬液，留下组织落户件的。毛皮疗法稀释细胞悬液与Neurobasal/B27完全培养基10-12毫升。 </li><li>拌匀，加入10μL细胞悬液490μl50X 1.5毫升的Eppendorf管中的计数解决方案（ 见表三 ）的稀释细胞计数。 </li><li>对PDL涂层板板细胞密度在5.0×10 4 /厘米2。如果要执行核转，我们建议在高浓度（8-10×10 4 /厘米2）电镀细胞。 </li><li>通常情况下，我们解剖实验9-10％的胎儿，约13×10 6个神经元E17的胎儿从每个派生。如果需要更多的胚胎，确保整个过程不会持续了两个多小时。 </li><li>如果需要隔离，24小时后，10微米胞嘧啶β-D-阿糖（阿糖胞苷）可添加到每一道菜，为了防止神经胶质增生。然而，这一步是不需要自Neurobasal/B27介质，抑制神经胶质增生每制造商的建议（Invitrogen公司/ Gibco公司）。 </li><li>神经元可用于经过4-5天， 在体外实验中，虽然确切时间取决于所需的分化阶段。我们有没有生存的显着减少（ 图1）长达4周的培养神经元。 </li><li>对于扩展的培养，每周更换培养液，用新鲜配制Neurobasal/B27完全培养基。 </li></ol> 5。代表结果玻璃玻片上培养的神经细胞受到免疫组化。 图1显示了固定后5天在文化和反MAP-2抗体，表明神经元突起immunolabeled皮质神经元的典型形象。 图2显示了文化3周后大鼠海马神经元的形象代表。一个完全分化的细胞，神经元形态，突出了MAP-2我mmunolabeling（MAP-2神经元的标记，AP-20单克隆抗体的克隆小鼠，基因TEX，尔湾，加利福尼亚州），按照标准的程序，如先前所述1。尼康的Eclipse E400直立荧光配备鄂西水族相机（Qimaging），机动Z轴，SlideBook5收购/反卷积软件（智能影像创新公司，丹佛，CO）显微镜图像的可视化。一系列每一个人的图片三维图像解卷积到一个二维图像和调整的信号，切断附近的最大强度，提高分辨率来解决。 图3显示了神经细胞的纯度。蛋白裂解DIV7大鼠神经细胞（CTX），并从人类神经母细胞瘤（GBM）的情况下获得的。正如预期的那样，是强烈的神经元蛋白MAP-2和星形细胞标志物GFAP阴性阳性神经细胞裂解的大紫荆勋章的蛋白质裂解为负f或地图-2阳性GFAP的。 虽然在我们的协议，我们已经使用几年解剖和冲洗介质的Hibernateé，最近我们已经探索它的一个额外和非常实用的使用，保存为进一步利用脑组织。 图4说明5（DIV5） 在体外天保持在4°C，在Hibernateé+ B27的一个星期后，他们从胚胎的原始解剖皮质分离的大鼠皮层神经元的文化。如前所述，PDL和层粘连蛋白涂在玻璃室幻灯片神经元接种。获取的图像解卷积使用SlideBook5收购/反卷积软件如上所述（ 图2）。 <img alt="图1" src="/files/ftp_upload/3965/3965fig1.jpg" /> 图1。（Amaxa，龙沙，Walkersville，博士）pmaxGFP nucleofected与地图immunolabeled的皮质神经元的形象代表-2抗体，红色。原放大倍率100倍。 <img alt="图2" src="/files/ftp_upload/3965/3965fig2.jpg" /> 图2显示地图-2免疫标记，海马神经元培养3周后，红色的代表形象。 DAPI染色，在蓝色的，显示出细胞的细胞核。原来的40倍放大倍率。 <img alt="图3" src="/files/ftp_upload/3965/3965fig3.jpg" /> 图3 Western blot分析显示神经细胞培养的纯度。 30微克的大鼠神经细胞和人类的大紫荆勋章蛋白裂解分离，电泳和Western blot分析，按照标准程序1。抗MAP-2是从细胞信号转导（丹弗斯，马萨诸塞州）兔多克隆抗GFAP抗体单克隆鼠标Chemicon公司（Millipore公司，比尔里卡，马萨诸塞州），小鼠单克隆抗-GRB2抗体（BD转导实验室火花，MD）。 GRB2被用作装载控制。 </P&gt; <img alt="图4" src="/files/ftp_upload/3965/3965fig4.jpg" /> 图4 在体外 5天。代表照片（DIV5）留在皮质大鼠皮质神经元的Hibernate E +在4°C一周后，他们解剖B27的一个。 A）的双室玻璃幻灯片上培养的神经元相衬。原放大倍率20X。二）免疫荧光显示神经元的过程中MAP-2的表达，在绿色文化为星形细胞标记物GFAP阴性。 DAPI染色，在蓝色，表明细胞的细胞核。原来的40倍放大倍率。

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没有利益冲突的声明。

大鼠海马和皮层神经元的解剖和文化在这里介绍的方法，允许使用在化学上定义的介质（ 图3）增长了近纯神经细胞进行实验。虽然近纯无血清培养基中培养神经元的协议已先前所述的2,3,4，也有我们的方法中作出了重要的变化。不同于传统的协议（即银行家等）5，我们已取代TrypLE快速，更温和的分解酶胰蛋白酶。我们也省略了两个步骤，这可能会影响神经细胞的完整性:皮质或海马切碎酶解前和使用DNA酶。另外，在休眠é胚胎解剖（步骤4.2），帮助维护可行的和健康的细胞。 Hibernate的E是一种利用神经组织或细胞在维护环境的二氧化碳含量的营养培养基（Invitrogen公司，生命科技，大ISL，纽约）。最初制定的航运孤立的脑区（海马，纹状体和皮层），休眠é介质可用于保持大脑可行的，在神经组织​​的隔离或显微镜，电或流式细胞仪在保持健康的神经元。事实上， 图4显示了两玻璃室幻灯片5天培养的大鼠皮层神经元的代表图片。在此之前他们的处理，皮质分别在Hibernate保存在4°C E + B27的黑暗一周后，他们从E17的胚胎解剖。有趣的是，从几天保持在4°C的组织分离的神经元形态与那些被分离出胚胎解剖的同一天。具有隔离在不同时间解剖后的胚胎神经细胞的选项，可以规划实验时，因此，在充分利用的时间怀孕的动物，是非常有帮助。 应该被mentione的ð从E13到E17的胚胎，我们的协议有效运作，但它从来没有被新生或成年啮齿动物测试。它也为人类胎儿的神经元（未发表资料）的隔离和文化工作非常出色。 这里介绍的方法分离的细胞可以镀在组织培养皿实验，需要收获大量细胞的RNA / DNA或蛋白质的提取。例子包括一些化合物或多肽，如在我们的工作，对HIV-Tat蛋白1，神经元的处理。生长因子介导的信号通路，还可以进行调查或RNA可用于纯化基因表达的miRNA分析阵列6。 此外，电镀前的神经元的核转染提供了一个工具来探讨分子或生长条件，影响神经细胞的分化1。其他应用还包括2000年通过脂质体转染分化的神经元（Invitrogen，卡尔斯巴德，加利福尼亚州）1。虽然转染效率是相当低的核转染相比，它是适用于像检测非常敏感的荧光素酶报告基因检测或电生理研究。此外，玻璃玻片上培养的神经细胞受到免疫组化（ 图1和图4B）1。最后，我们成功地遵循了这一协议，从小鼠胚胎的神经祖细胞7，游离在修改的最后一步，在孤立的神经祖细胞定义的介质中培养，如先前所述7,8。总体而言，这个简单的方法有多种应用和容易为不需要胶质细胞支持神经细胞的研究提供了神经。但是，胶质细胞条件培养基联合培养或胶质源性可以加入到这些神经细胞检查9胶质细胞诱导突触的形成机制如由Pfrieger和Ba描述研究，RRES 10。

<table border="1"><tbody><tr><td> 试剂 </td><td> 浓度 </td></tr><tr><td>的Neurobasal </td><td> 98％ </td></tr><tr><td> B27的</td><td> 2％ </td></tr><tr><td> glutamax </td><td> 0.5毫米</td></tr></tbody></table> 表一Neurobasal/B27完全培养基 。 <table border="1"><tbody><tr><td> 试剂 </td><td> 浓度 </td></tr><tr><td>葡萄糖</td><td> 16毫米</td></tr><tr><td>蔗糖</td><td> 22毫米</td></tr><tr><td>肝素钠</td><td> 10毫米</td></tr><tr><td>氯化钠</td><td> 160毫米</td></tr><tr><td>氯化钾</td><td> 5毫米</td></tr><tr><td>的Na 2 HPO 4 </td><td> 1毫米</td></tr><tr><td> KH 2 PO 2 </td><td> 0.22毫米</td></tr><tr><td>庆大霉素</td><td> 50微克/毫升</td></TR&gt; <tr><td> fungizone </td><td> 250毫微克/毫升</td></tr><tr><td> pH值</td><td> 7.4 </td></tr><tr><td>渗透压</td><td> 320-330 mOsm </td></tr></tbody></table> 表二。夹层介质 。 <table border="1"><tbody><tr><td> 试剂 </td><td> 量（μl） </td></tr><tr><td> Neurobasal/B27完整的介质</td><td> 240 </td></tr><tr><td>台盼蓝染色0.4％ </td><td> 250 </td></tr><tr><td>总</td><td> 490 </td></tr></tbody></table> 表三。 50X计算解决方案 。 <table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 猫。数 </td></tr><tr><td> Hibernate的é </td><td> brainbits </td><td> 767171 </td></tr><tr><td>的Neurobasal </td><td> Gibco公司，Invitrogen公司</td><td> 21103-049 </td></tr><tr><td> B27的</td><td> Gibco公司，Invitrogen公司</td><td> 17504-044 </td></tr><tr><td> fungizone </td><td> Gibco公司，Invitrogen公司</td><td> 15290-018 </td></tr><tr><td>硫酸庆大霉素</td><td> Sigma Aldrich公司</td><td> G1264 </td></tr><tr><td> glutamax 200毫米</td><td> Gibco公司，Invitrogen公司</td><td> 35050 </td></tr><tr><td> TrypLE快递W / O酚红</td><td> Gibco公司，Invitrogen公司</td><td> 12604 </td></tr><tr><td>胞嘧啶β-D-阿糖盐酸盐</td><td> Sigma Aldrich公司</td><td> C6645 </td></tr><tr><td>聚-D-赖氨酸</td><td> Sigma Aldrich公司</td><td> P6407 </td></tr><tr><td>层粘连蛋白1毫克/毫升</td><td> Millipore公司</td><td> CC095 </td></tr><tr><td>肝素钠</td><td> Sigma Aldrich公司</td><td> H3375 </td></tr><tr><td>台盼蓝染色0.4％ </td><td> Gibco公司，Invitrogen公司</td><td> 15250 </td></tr></tbody></表&gt; 表四。特定的试剂 。 <table border="1"><tbody><tr><td> 设备 </td><td> 公司 </td><td> 猫。数 </td></tr><tr><td>立体显微镜</td><td>奥林巴斯</td><td> SZ61 </td></tr><tr><td>大钳</td><td>福斯特</td><td> 11022-14 </td></tr><tr><td>细尖镊子</td><td>莫里亚</td><td> MC40B </td></tr><tr><td>微精尖镊子</td><td>莫里亚</td><td> MC31 </td></tr><tr><td>剃刀般锋利的剪刀</td><td> roboz </td><td> RS-6820 </td></tr><tr><td>微解剖剪刀</td><td>福斯特</td><td> 91460-11 </td></tr><tr><td>微解剖弯剪刀</td><td>福斯特</td><td> 14067-11 </td></tr><tr><td>玻璃2玻片</td><td>实验室康</td><td> 154461 </td></tr><tr><td> 60毫米菜</td&gt; <td>屋宇署猎鹰</td><td> 353002 </td></tr><tr><td> 100毫米菜</td><td>康宁</td><td> 430167 </td></tr><tr><td> 15毫升管</td><td>屋宇署猎鹰</td><td> 352099 </td></tr><tr><td> 1.5毫升冷冻管瓶</td><td> NUNC </td><td> 375353 </td></tr></tbody></table> 表五，特殊设备 。

isolation and culture of rat embryonic neural cells: a quick protocol

我们描述了一个快速的方法分离和培养E15-17大鼠胚胎海马或皮层神经元。程序可以成功地应用于老鼠和人类的初级神经元和神经祖细胞的分离。分离神经元无血清培养基均保持在几个星期。这些文化可用于核转染，免疫细胞化学，核酸制备，以及电。年老的神经细胞，也可以慢病毒转导效率，有效地减少，磷酸钙或脂质的方法，如脂质体转染。

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

我们描述了一个快速的方法分离和培养的海马和皮层神经细胞从老鼠胚胎。该协议允许在几乎纯神经细胞需要我们进行实验。

大鼠胚胎神经细胞的分离和培养:一个快速议定书"

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

isolation-and-culture-of-rat-embryonic-neural-cells-a-quick-protocol

Nanyang Technological University

Research

JoVE Journal

Neuroscience

38.1K 次观看.  LSU Health Sciences Center - New Orleans. 我们描述了一个快速的方法分离和培养的海马和皮层神经细胞从老鼠胚胎。该协议允许在几乎纯神经细胞需要我们进行实验。

视频: Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人力蕈状味觉​​乳头细胞的分离和培养

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...