研究 Spagnoli实验室。是由亥姆霍兹联合会，FP7-IRG-2008-ENDOPANC补助金及ERC-2009年开始肝胰腺津贴。

已经适应这里描述的协议，从技术最初描述的波斯 ​​富街和松弛9共聚焦显微镜和优化。 1。涂层的玻璃底培养皿应进行以下步骤，在无菌条件下在层流罩中。 <ol><li>胰腺外植体培养在35毫米的培养皿中，用直径为20毫米的玻璃微孔底部公司（ 例如 MatTek Corporation）。使用一个玻璃底菜，每外植体培养的整个期间的文化。在一天前的胰芽，涂在玻璃底部微孔纤维连接蛋白的解剖和隔离。 </li><li>细胞培养级水稀释无菌纤连蛋白在50μg/ ml的最终浓度，并添加最小体积的（约150微升）到20毫米的微孔的MatTek板覆盖整个玻璃表面上（ 图1E）。将菜在4℃冰箱中过夜培养。 </li><li>在当天的清扫，吸出纤维连接蛋白，细胞培养级水冲洗微孔，增加的最小容积150微升BME（基础培养基鹰）培养基青/链霉素（1％），谷氨酰胺（1％ ）和50微克/毫升庆大霉素[解剖中等]。在层流罩板的外植体，直到准备离开涂层的菜肴。 </li></ol>如果不是所有的纤连蛋白涂覆的菜肴被使用，它们可以被存储长达1周在4℃下不要让菜干，夹层中填充它们，并将其放置在冰箱中。 除了 ​​对纤连蛋白，胰腺外植体已经成功培养在各种基材上，例如为9层粘连蛋白，基质胶10,11或微孔膜12。由于其对分枝的效果，我们使用作为底物的纤维连接蛋白9。 2。解剖的Dorsal的胰芽从E11.5 - E12.5小鼠胚胎<ol><li>定时怀孕的雌性小鼠在胚胎天（E），11.5或12.5的牺牲和胚胎收集使用解剖仪器，以前用70％乙醇清洗。将解剖子宫在含有补充有50微克/毫升庆大霉素的冰冷的PBS的10-cm的板。 </li><li>体视显微镜下成单独的胚胎段，用小剪刀或杜蒙钳轻轻剥离远离子宫的肌肉和删除单个胚胎，蜕膜组织所包围，从上面的，独立的子宫照明。使用标准的胚胎解剖技术13，揭露的胚胎和删除卵黄囊和羊膜。接着，取出胚胎（上方的肝脏）和尾部区域的上半身。这将允许内部器官（ 图1B）的曝光。中期体胚胎转移到10厘米的板含有冰冷BME夹层介质。 </li><li>现在从下面只使用透，可视化和背胰芽体视显微镜下解剖。轻轻地，打开中期的胚胎体的横向切口，用血管钳。找到胃和脾。背胰芽横向安装到后方区域的胃（ 图1C）。使用蒙特镊子，解剖走背胰芽从邻近结构，如胃和脾脏，但留下了一些间充质细胞周围的上皮细胞（ 图1D）。使用玻璃巴斯德吸管，转移成一个35毫米的培养皿中含有冷的BME夹层培养基后，用10％胎牛血清的[培养基]孤立芽。重复上述步骤，直到所有胰腺被隔离。如果胰腺不同基因型是孤立的，让他们分开使用Nunc公司4孔板。 </li></ol> 3。胰腺外植体的电镀和文化最终平台ING的外植体进行组织培养室和/或在邻近的组织培养箱培养，以尽量减少的物理运动。 <ol><li>在层流罩中无菌条件下，取代的BME夹层中等，纤连蛋白涂覆MatTek菜肴预先温热在37〜150微升的BME培养基℃。 </li><li>小心，胰腺外植体转移到的涂层MatTek菜肴（每道菜的一个外植体）长期培养使用玻璃巴斯德吸管。要确保文化的传播过程中，外植体周围的间充质可以稍微撕裂，如果必要的话，用细针。 </li><li>轻轻地，放置在培养的组织培养箱（37℃，5％CO 2）的几个小时内，让外植体附加到玻璃底微孔。一旦它们所连接，填补MatTek菜肴预先温热的BME培养基中的1.5毫升〜2毫升在37℃下夹层的天被称为第0天。 </li><li>曼ipulation的胰腺外植体培养的是，在层流罩中无菌条件下进行的。 BME培养基改为隔日，，除非实验要求规定不同的时间（ 如培养的外植体，用化学药剂）。的外植体可以在此条件下培养5天至一周。 </li></ol> 4。整个安装胰腺外植体免疫荧光染色协议所有步骤的的免疫荧光协议（固定成像）进行在同一MatTek的一道菜，产生更好的图像比塑料底的菜。固定后，胰腺外植体的培养并不需要保持在无菌条件下。 <ol><li>吸BME培养基，2毫升1X PBS清洗一次外植体。 </li><li>外植体固定，取代用2ml冰冷的4％多聚甲醛（PFA）的PBS的培养皿放置在冰上20分钟。轻轻地从时间旋流板时间，但要避免摇摆平台，为外植体分离。 </li><li>移除PFA和用2毫升，每次10分钟，在室温（RT）的1X PBS冲洗外植体三次。 </li><li>块用2毫升封闭溶液（1X PBS + 0.1％的Triton-X + 3％驴血清）为至少30分钟，在RT。 </li><li>一次抗体，在封闭溶液稀释，并〜150微升的稀释液添加到20毫米的微孔的MatTek板覆盖，在4℃下培养过夜的整个外植体表面直接为了减少体力的运动，加入一抗外植体直接在冷室（4°C）。为了防止水分蒸发，增加抗体之前，将MatTek板在潮湿的室内。 </li><li>第二天，除去抗体溶液，并用2毫升的1X PBS洗涤三次+ 0.1％Tween-20的（PBT）的为每个在RT 30分钟。 </li><li>根据制造商的建议稀释的二抗封闭液，我们使用的是驴的Alexa Fluor染料二抗机构在1:750稀释，并添加150微升稀释液直接到20毫米的微孔的MatTek板，覆盖整个外植体表面，在室温下培养1-2小时在黑暗中。对于核counterstaining，包括赫斯特连同第二抗体稀释液。为了防止水分蒸发，增加抗体之前，将MatTek板在潮湿的室内。 </li><li>洗涤三次，用2毫升的1X PBS + 0.1％Tween-20的（PBT）的30分钟，在RT。 </li><li>在安装之前，用1X PBS清洗一次，以消除吐温20。通过添加1滴水溶液安装介质（带防褪色）到20毫米的微孔的MatTek板安装。轻轻地，一个盖玻片覆盖的外植体。避免气泡。 </li><li> MatTek板的外植体对可以根据标准相衬显微镜或共聚焦显微镜，观察取决于实验的目标上。共聚焦显微镜分析中，我们使用了蔡司LSM 700倒置​​的设置。设置适当的激光器的荧光基团我们编辑。选择一个目标，将图像的胰腺外植体所需的区域。阿10X的水浸入的目标是合适的，以保持整个外植体中的视场。 40倍的水或油浸泡的目标是合适的专注于特定区域的胰腺外植体更高的分辨率。的外植体可以是光学切片，然后所取得的Z-堆栈在3D重建，使用适当的软件。 </li></ol> 5。胰腺外植体活细胞成像协议<ol><li>环境室设置：[使用环境的外壳和一个小型孵化室温度和CO 2控制]坐落在显微镜载物台的。正确培养的胚胎胰腺需要一个稳定的温度为37℃和湿润的5％CO 2的受控气氛。来自多个供应商提供合适的房间是 （ 例如 ，蔡司，尼康，奥林巴斯）。组装的加热箱中，并把它在一个吨至少1小时开始成像之前，以允许设备预热并平衡至5％的CO 2含量。 </li><li>我们对于时间的推移成像胰腺外植体培养，使用蔡司LSM 700倒置​​显微镜。激光器和目标选择，见4.10点。 </li><li>让胰腺外植体附着到玻璃底的菜才开始实时成像。我们通常开始的第1天]进行实时成像。 在层流罩中，吸液和替换的外植体的培养基用新鲜的BME培养基从最佳营养条件。 </li><li>轻轻的外植体转移到显微镜室和放置的MatTek的培养皿中孵化室，试样架共聚焦显微镜。 </li><li>准备的目标（ 例如，油或水介质添加根据浸没物镜的选择），并集中在感兴趣的区域中的外植体。正确关闭的环境室。为了防止蒸发在时间的推移中采集与浸水目标，代替水，它是推荐使用粘性的浸没流体是市售的（ 例如蔡司Immersol W）。 </li><li>设置激光强度栈Z-采集的参数和时间间隔（见故障排除还讨论）。开始实验。 </li><li>成像后，出口ž堆栈和所有的时间点，并分析他们使用合适的软件（ 如 ImageJ的，Volocity，Imaris，惠更斯）。 </li></ol> 6。代表性的成果背胰芽（连同周围的间充质细胞）进行了解剖，从小鼠胚胎E11.5和E12.5和直接镀在玻璃底培养皿（ 图1）。血管是存在于周围的上皮细胞的间充质细胞，可以被可视化的内皮标志物PECAM 12（数据未示出）通过免疫染色。经过两天的文化，胰腺外植体UNDerwent显着增长，并开始组织起来，支上皮结构，其细胞核呈阳性的胰腺转录因子，PDX1（ 图2A-C）。平均胰腺外植体在第3天的文化Z-厚度为60-80微米。外植体的表面和体积的测量可以使用适当的软件（ 例如惠更斯）。形态学和免疫荧光分析表明，胰腺外植体培养，使用此协议的概括在体内的胰腺癌早期分化和形态发生的事件5,10,11,14（ 图2）。胰腺外植体天4文化上接受了典型的“尖和主干”偏析上皮8，显示CPA1 +祖细胞在上皮分支（ 图2C）和分化胰岛素+和胰高血糖素+细胞簇后备箱内组织的尖端（ 图2D ）。此外，胰腺炎Ç显示正确的上皮细胞在体外培养细胞骨架组织和细胞极性，心尖部本地化的F-肌动蛋白（ 图2E，绿色）和b1整合在基膜（ 图2E，红色）。 为研究胰腺分支的实时，胚胎胰腺外植体分离的双基色荧光灯记者的小鼠品系membrane-Tomato/membrane-Green 15（吨/毫克;在杰克逊实验室小鼠品系）（ 图3）。随时间推移电影万吨/ MG胰腺外植体在培养基中培养从静止帧， 如图3所示。本地化的MT荧光蛋白膜结构勾勒细胞形态及精细胞的过程，随着时间的推移，能够跟踪细胞重塑和迁移使分辨率。 经过12个小时的时间推移实验MT荧光信号仍然是可行的，DETEctable，即使它的强度降低，最有可能是由于光损伤（ 图3）。在讨论中讨论的技术解决方案，以避免或减少光损伤和光毒性。 <img alt="图1" src="/files/ftp_upload/3979/3979fig1.jpg" /> 图1。从E12.5小鼠胚胎背侧胰芽解剖示意图。 （A）明E12.5小鼠胚胎。胚胎的上半身（上方的肝脏）和尾部区域中除去隔离中期体（B）。切口红色虚线所示。 （C）进一步解剖的曝光结果，胃和十二指肠的区域（由虚线框表示）。背胰芽（见红色虚线圆圈）是在该基地的胃和脾原基。用巴斯德移液管转移到玻璃底培养皿含有BME培养基中的微孔（E解剖背胰芽（D）的的F）。缩写：STO（胃），SP（脾），DP（背胰芽），双核（十二指肠）。比例尺，1000微米（A，C）。 <img alt="图2" src="/files/ftp_upload/3979/3979fig2.jpg" /> 图2。整个安装共聚焦免疫荧光分析胰腺外植体培养。 （A）明图像的胰腺外植体对体外培养的第1天，第2天的。红色虚线显示的上皮分支的开始。 （B）免疫组化三维重建整个安装PDX1在第3天胰腺文化。胰腺外植体显示管PDX1 +细胞48小时的文化进行广泛的分支。 （C）所有安装免疫的第3天胰腺外植体抗体基底膜标记E-cadherin蛋白（ECAD），​​尖祖标记，羧肽酶1（CPA1）和磷酸化组蛋白H3（pHH3）。大多数的有丝分裂活性pHH3 +细胞共定位在周与CPA1 +细胞的提示。虚线升国际核事件分级小费的分支机构。 （D）整体安装的的免疫内分泌的血统标志物，胰岛素（Ins）和胰高血糖素（Gluca）在第3天。黄色箭头指示集群的INS + Gluca +细胞。插入（D&#39;）中，高倍率的内分泌细胞集群之一。 （E）所有安装的第3天胰腺外植体免疫PDX1，β1整合素（β1int）和F-肌动蛋白（事实）。穿插其中PDX1阳性上皮细胞的间充质细胞（星号）。在C，D和E所示的画像是单一的共焦光学部分的Z系列。比例尺，100微米（A，B），50微米（CE）。 <img alt="图3" src="/files/ftp_upload/3979/3979fig3.jpg" /> 图3。 体外培养的胰腺外植体活细胞成像的代表。 （AD）的帧从一个MT / MG小鼠转基因胚胎胰腺外植体随时间推移电影。成像时示出在“小时：分钟。第1天开始，外植体成像。 Z-堆栈的图像被收购总量的15小时，每12分钟为一个40倍油浸物镜。虚线表示翻倒的分行及上皮细胞和间充质之间的边界。比例尺，50微米。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+division+in+branching+morphogenesis+and+differentiation+of+the+embryonic+pancreas.">Role of cell division in branching morphogenesis and differentiation of the embryonic pancreas.</a> Int. J. Dev. Biol. 44, 791-796 (2000).</li><li>Muzumdar, M., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

没有利益冲突的声明。

胰腺癌的命运一旦被指定，进行广泛的胰腺祖细胞的增殖，分化和形态，最终形成一个成熟的，功能性的器官2,4。目前，如何分支发生在胰腺和它是如何连接到祖细胞增殖和分化在很大程度上是未知的。胰腺外植体培养代表阐明这些过程的体外 5,9,11一个理想的系统。与早期胰腺癌芽体外植活细胞成像相结合，可以得到一个清晰的画面在胚胎胰腺形态发生过程中发生?...

<table border="1"><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>抗体： 羧肽酶 E-钙粘蛋白 F-肌动蛋白 胰高血糖素 胰岛素 β1整合素 PDX1 PDX1 磷酸化组蛋白H3 </td><td> ABD Serotec公司 Invitrogen公司 分子探针 ImmunoStar 密理博 密理博 Abcam公司 杂交瘤细胞银行 细胞信号</td><td> 1810-0006 13-1900 A-12373 20076 4011-01 MAB1997 ab47267 F109-D12 9706 </td><td></td></tr><tr><td>的基础培养基鹰（BME） </td><td>西格玛</td><td> B1522-500ML </td><td>存放在无菌条件下</td></tr><tr><td>细胞培养级水</td><td> PAA </td><td> S15-012 </tD&gt; <td>存放在无菌条件下</td></tr><tr><td> （玻璃底培养皿），35毫米</td><td> MatTek公司</td><td> P35G-0-20-C </td><td></td></tr><tr><td>驴血清</td><td> Chemicon公司</td><td> S30-100毫升的</td><td></td></tr><tr><td>胎牛血清的黄金</td><td> PAA </td><td> A15-151 </td><td>存放在无菌条件下</td></tr><tr><td>纤维连接蛋白</td><td> Invitrogen公司</td><td> 330100-8 </td><td>股票溶胶。 1 mg / ml的细胞培养级水</td></tr><tr><td>庆大霉素</td><td> Invitrogen公司</td><td> 15750-037 </td><td>存放在无菌条件下</td></tr><tr><td>谷氨酰胺</td><td> Invitrogen公司</td><td> 25030-024 </td><td>存放在无菌条件下</td></tr><tr><td> 4孔Multidishes的</td><td> Nunc公司</td><td> 176740 </td><td></td></tr><tr><td>显微镜： 倒立共轭焦显微镜（LSM 700） 体视显微镜（愉景V12） </td><td>蔡司 蔡司</td><td></td><td>目标： 复消色差C-10X / 0.45 W M27（work.区1.8毫米，成像深度〜100毫米），C-的复消40X / 1.2 W科尔M27（work.区0.28毫米，〜显像深度50微米） 透照从上方从下面和光纤照明</td></tr><tr><td>多聚甲醛</td><td>罗斯</td><td> 0335.3 </td><td>原液20％ </td></tr><tr><td>巴斯德移液管（玻璃），150毫米</td><td> VWR </td><td> HECH567 / 1 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> PAA </td><td> P11-010 </td><td>保持在无菌Çonditions </td></tr><tr><td>培养皿中，60毫米</td><td>格雷纳生物之一</td><td> 628102 </td><td></td></tr><tr><td>培养皿中，35毫米</td><td>格雷纳生物之一</td><td> 627161 </td><td></td></tr><tr><td> 1X PBS，pH7.4的</td><td> PAA </td><td> H15-002 </td><td>存放在无菌条件下</td></tr><tr><td>春风似剪刀8毫米刀片弯曲</td><td>精细科学工具</td><td> 15023-10 </td><td></td></tr><tr><td>的Triton-X100 </td><td>罗斯</td><td> 3051.3 </td><td></td></tr><tr><td>钟表foreceps杜蒙第5 </td><td>罗斯</td><td> K342.1 </td><td></td></tr></table>

a system for ex vivo culturing of embryonic pancreas

胰腺控制我们的身体重要的功能，包括消化酶和调节血糖水平的生产。虽然在过去的十年中，许多研究都促成了坚实的基础，为了解胰腺器官，重要的差距仍然存在在我们的知识的早期胰腺形成2。将提供一个完整的了解，这些早期事件的洞察这个机构的发展，也成为目标的胰腺疑难杂症，如糖尿病和胰腺癌。最后，这些信息将产生发展的背景下，糖尿病的细胞替代疗法的蓝图。 在胚胎发育过程中，胰腺源于不同的胚胎副产物的背，腹肠胚层在胚胎天（E）9.5在小鼠胚胎3,4。这两种副产物向外翻固体上皮细胞周围的间充质升芽，进行增殖，分支和分化的产生一个完全成熟的器官2,5,6。最近的证据表明，生长和分化的胰腺细胞谱系，包括产生胰岛素的β细胞，依赖于适当的组织架构，上皮重构和细胞定位分支胰腺上皮内7,8。然而，如何分支形态发生与细胞增殖和分化的胰腺中的协调在很大程度上是未知的。这部分是由于这些发育过程的了解，目前几乎完全依赖于固定标本的分析，而形态发生的事件是高度动态的。 在这里，我们提出一个用于解剖的方法和培养小鼠胚胎胰腺芽体外玻璃 ​​底的菜肴，可以直接可视化的胰腺（ 图1）。这个邪教余吕杏茜系统是理想的设计，激光扫描共聚焦显微镜，特别是活细胞成像。胰腺外植体可以不仅从野生型小鼠胚胎制备，但也从遗传工程小鼠品系（ 例如转基因或基因敲除），允许实时的突变表型的研究。此外， 体外培养系统是值得研究的化学化合物对胰腺发育的影响，从而获得定量数据的增殖和生长，延伸，分支，tubulogenesis和分化。总之，的体外胰腺外植体培养法结合高分辨率成像的发展提供了一个强大的平台，观察形态发生和分化的事件发生，因为他们在发育中的小鼠胚胎。

Watch this Scientific Journal Video about A System for ex vivo Culturing of Embryonic Pancreas at JoVE.com

A System for ex vivo Culturing of Embryonic Pancreas

在这里，我们描述了一个小鼠胚胎胰腺分离，培养和操纵方法。这是一个极好的<em&gt;体外</em系统为研究胰腺发育的各个方面，包括形态，分化和生长。胰芽外植体可以培养数天，并用于在一个范围内的不同的应用程序，包括整体安装免疫荧光和实时成像。

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

a-system-for-ex-vivo-culturing-of-embryonic-pancreas

Research

JoVE Journal

Biology

A System for ex vivo Culturing of Embryonic Pancreas

13.6K 次观看.  Max-Delbrück-Center for Molecular Medicine. 在这里，我们描述了一个小鼠胚胎胰腺分离，培养和操纵方法。这是一个极好的体外

视频: A System for ex vivo Culturing of Embryonic Pancreas

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

一个培养人关节软骨细胞的三维系统在滑液

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

体外胰腺器官从分散的小鼠胚胎祖细胞

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Methods Article

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