The work was supported by grants from the Universit&#233; catholique de Louvain (Action de recherch&#233; concert&#233;es) and the Fund for Scientific Medical Research (F.R.S.-FNRS, Belgium). A.-S.D. is a doctoral fellow from T&#233;l&#233;vie, A.-C.H. held a fellowship from the Fonds pour la formation &#224; la Recherche dans l'Industrie et dans l'Agriculture (Belgium), M.V. is supported by the de Duve Institute and C.E.P. is a senior research associate of the F.R.S.-FNRS (Belgium).

小鼠按照实验动物管理大学动物福利委员会的原则，提出和处理。所有的程序和协议已获本委员会。 1，显微塑料培养室的涂层注意：执行以下所有在层流通风橱的无菌条件下的步骤。这两种类型的涂层在功能上是等价的。 <ol><li>外套塑料培养室与纤维连接蛋白的前一天甲状腺组织的隔离： <ol><li>稀释无菌纤连蛋白在M199培养基50微克/毫升的最终浓度补充有100单位/毫升青霉素，100微克/毫升链霉素，0.25微克/毫升两性霉素B和2mM谷氨酰胺12。 </li><li>盖井200微升稀释纤维连接蛋白，并在4℃冰箱孵育过夜。 </li><li>在解剖当天，吸出纤连蛋白，与M1冲洗各孔一次99，并添加200微升M199的补充有抗生素，谷氨酰胺和10％胎牛血清的最低量。 </li><li>留在孵化器的涂层室，直到准备板的外植体。 </li></ol></li><li>外套塑料培养室与I型胶原2小时的甲状腺组织中分离之前： <ol><li>稀释无菌I型胶原蛋白，以在10mM盐酸50μg/ ml的终浓度。注：置于冰上胶原蛋白原液。 </li><li>覆盖所述井用200微升稀释的I型胶原并孵育2小时，在室温（RT）。 </li><li>吸出I型胶原溶液的类型，漂洗各孔一次，M199，并添加200微升M199的补充有抗生素，谷氨酰胺和10％胎牛血清的最低量。 </li><li>留在孵化器的涂层室，直到准备板的外植体。 </li></ol></li></ol>甲状腺含Anlagen的组织2。解剖（E12.5）或甲状腺叶（E13.5-E14.5） 注：使用干净的解剖工具和Petri /培养板。下面描述的一般步骤和切口都是相同的E12.5和E14.5胚胎。 <ol><li>牺牲定时怀孕的雌性小鼠在所需的胚胎（五）天（从E12.5到E14.5），并使用解剖仪器切除子宫。 </li><li>将切开子宫在含有HBSS 10cm的板。 </li><li>握住钳子子宫的一个末端，并用小剪刀逐步释放所有打开的胚胎在子宫。 </li><li>从用剪刀胎盘中分离胚胎和转移他们的胚外膜的胚胎在含有HBSS一个新的10cm平板。 </li><li>删除卵黄囊和羊膜。注：从这一点来说，用下从下方透体视显微镜工作;增加放大倍数与解剖步骤。使用钨针在玻璃毛细管作为解剖工具。 </li><li>使用诸如针刀叉，去除头的上半部分，包括上颚和耳朵（ 图1A，沿切）。 </li><li>按住胚胎在它的后面，并先删除一个额外的一块神经管（ 图1A，沿乙切），胚胎，然后下部，刚下过前四肢和心脏上方（ 图1B）。 </li><li>含有转舌（以定向组织）和颈部区域中包含的HBSS一个新的10cm平板胚胎切片。 </li><li>除去神经管和组织后方的食道/气管（ 图1C，沿着切割）。 </li><li>轻轻部组织切片沿舌（ 图1C，沿着B和B&#39;切割）的两侧。 </li><li>找到食道和气管，在舌头的延长，并剖析了舌头，留下杓状肿胀（ 图1D，沿切），并在BOT组织食道/气管（ 图1D，沿着B和B&#39;切割）h的侧面。 </li><li>取出食管，并放置气管，其腹面朝上。 </li><li>解剖掉不想要的组织如胸腺（ 图1E）和所有位于横向于咽弓的动脉组织。为E13.5或E14.5胚胎，可视化的甲状腺瓣上气管的每一侧（ 图1F;红色虚线）和从气管进一步解剖瓣。 </li><li>所述分离E12.5气管区，或E13.5-E14.5甲状腺瓣转移到含有补充了抗生素，谷氨酰胺和10％胎牛血清的预热的M199培养基在35 mm培养皿。使用预湿过滤嘴在P-200微量转移外植体。对于E12.5植，切尖的末端，以扩大开放。注：按常规，如果胚胎有相同的基因型，表现在所有的胚胎步骤6和7，Ñ​​9-11，最后12＆13。 </li></ol> 3，电镀甲状腺外植体与文化<ol><li>通过连续转印在三个孔24孔板中含有1ml预热的培养基中洗涤外植体（= M199补充有抗生素，谷氨酰胺和10％胎牛血清）。 </li><li>镀上涂层的显微塑料培养室甲状腺叶： <ol><li>从涂覆室抽吸M199培养基中。 </li><li>小心，转移甲状腺植成使用微量，过滤嘴棒的培养室。放置1外植体中的每个腔室的中心。 </li><li>除去多余的培养基，并将培养容器在组织培养箱（37℃，5％CO 2）2小时，让外植体附着于基质。 </li><li>轻轻地添加200至300μl的预热的培养基到每个孔中。 </li><li>在组织培养培养箱中孵育长期培养。 </li></ol></li><li> Ø电镀上semiporous滤波器F1气管地区或甲状腺叶： <ol><li>填充330微升预热的培养基中所需要的24孔板的孔的数量。 </li><li>过滤器的地方（ 例如 0.4微米的微孔过滤器）的介质上，同时注意避免下面的文化过滤器气泡俘获。 </li><li>小心，使用微量过滤和技巧，转移甲状腺外植体用介质的最小体积到过滤器的中心（最大4/filter）。 </li><li>去除多余的介质和空间用钨针外植体。 </li><li>在组织培养培养箱中孵育长期培养。注：更改文化层流罩下，隔日媒体。培养的外植体长达7天。 </li></ol></li></ol> 4，全安装免疫荧光染色甲状腺植协议使用钨针或打跑生长在过滤器外植体（下面的步骤2后）细镊子和转让在24孔板中的所有步骤，除了初级（步骤5）和二级（第7步）抗体孵育（96孔板）。有关显微镜的塑料腔贴壁培养的外植体，进行在培养室中的所有步骤。 <ol><li>吸出物的M199培养基中，并固定用冰冷的4％多聚甲醛（PFA），20分钟（500微升在24孔培养皿和300微升在显微镜载玻片）的外植体。 </li><li>在RT;在Tris缓冲盐水用Triton洗2×10分钟（50毫摩尔Tris盐酸pH为7.5，150 mM氯化钠，0.1％的Triton X-100的TBST）。 </li><li>立即处理的外植体或储存在-20°C。注意：对于长期储存于-20℃，通过逐渐增加甲醇浓度在TBST达到100％甲醇脱水甲醇外植体。当准备继续与免疫染色（步骤4），通过逐渐加入TBST甲醇补充水分外植体。 </li><li>用封闭溶液（10％正常山羊血清在TBST中替换TBST）和孵化在海摇臂锯在室温30-60分钟。 </li><li>稀释第一抗体在阻断溶液（每个条件100-200微升）。从24孔板转移的“过滤器”外植体在96孔板中。培养外植体与在4℃下在海锯摇臂的第一抗体过夜</li><li>第二天，弃去第一抗体溶液，转移的“过滤器”植回在24孔板中，并用TBST上的海锯摇臂每个在室温45-60分钟冲洗至少5次。 </li><li>在含有正常山羊血清的1％的TBST稀释的第二抗体（ 例如山羊抗-X-的Alexa Fluor在1:2,000稀释）孵育的外植体与该稀释过夜上的海锯摇臂在4℃和在黑暗中。核复染，包括1微克/毫升双苯甲亚胺（Hoechst公司）与二级抗体溶液一起。 </li><li>第二天，弃去二级抗体溶液并用TBST上的海锯洗5倍摇杆，每个在室温45-60分钟。在清洗，保持在黑暗的外植体。 </li><li>执行在TBST轻轻摇动过夜，4°C黑暗额外洗。 </li><li>后缀在4％PFA的免疫染色的外植体在4℃下10分钟。 </li><li>用TBST洗涤2×10分钟。 </li><li>转移外植体或逐渐在甲醇100％长期储存于-20℃，或立即分析。为外植体上的过滤器中培养，并进行免疫染色在24孔板中，转印在显微镜室（ 例如 LabTek或Ibidi）用TBST或更好的荧光封固剂在分析前用多光子或共聚焦显微镜。 </li></ol> 5，提取RNA的甲状腺在无RNA酶的环境中工作。因此，使用核酸和核酸免费移液器吸头和污染物和RNA酶清洁两个板凳和设备。该协议是基于酚/氯仿，执行的第一步s（1至7）根据化学罩。 <ol><li>均质1甲状腺植或两个甲状腺叶用300微升的RNA提取液用一次性杵1.5 ml管。在样品之间，洗杵在2％SDS，然后在水，然后乙醇。 </li><li>继续添加100μl的RNA提取溶液中，涡旋10秒并在室温下孵育10分钟。 </li><li>涡旋30秒，加入20毫微克的tRNA到各样品中。 </li><li>涡旋混合1分钟，并加入80μl的氯仿中。 </li><li>涡流大​​力进行30秒，并培育15分钟，在室温。 </li><li>旋转15分钟，以19,000×g离心在冷藏（4℃）离心分离。 </li><li>小心转移含20微克的糖原作为一个共沉淀新的管无色的上层水相。 </li><li>涡流并添加200微升的100％异丙醇酒精。 </li><li>涡大力为15-30秒，并让RNA沉淀在-20℃下2-3小时，或在-80℃下1小时。 </li><li>旋转30分钟19,000XG在4℃下，并消除上清液。 </li><li>用450微升冷75％乙醇和涡流洗涤沉淀以去除沉淀。 </li><li>自旋为19,000 XG在4℃下10分钟，并消除上清液。 </li><li>干燥机罩5分钟下沉淀。 </li><li>重悬沉淀用10微升不含RNA酶的水中，并培育10分钟，在RT。 </li><li>检测RNA浓度，在-80℃下储存或反向转录基因表达分析。 </li></ol>

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The authors have nothing to disclose.

本文介绍了解剖，并以研究，更好地了解复杂的事件，导致甲状腺生成甲状腺培养外植体（E12.5）或叶（E14.5）的方法。由于甲状腺原基（中线原基和两个侧鳃体），和它们的小尺寸，E12.5组织局部喙向咽弓的动脉的片段，以及含有该气管，但不是食道的双重原点是显微切割。如图所示，为期四天之内，这种文化系统忠实地概括体内甲状腺的发展，因为：（i）本中线原基迁移双边和膨胀形成...

甲状腺是独立的上皮领域的集合，称为卵泡，通过密集的网络毛细血管内皮细胞所包围。该组织允许甲状腺功能：毛细血管内皮提供甲状腺与碘，所需的T3和T4激素的合成，并分发这些后期到全身。散落在毛囊和毛细血管之间，C-细胞产生的低血钙激素降钙素1。虽然成人甲状腺结构和功能是众所周知的，参与甲状腺胚胎发育（卵泡形成和分化）的细胞和分子机制远未清楚。 在胚胎发育，甲状腺祖起源的前肠内胚层的腹壁在胚胎一天增厚（中线原基）（e）在小鼠胚胎8.5，而C-细胞祖细胞起源于E11.5为液滴状突起（在鳃博死亡的第四咽囊2-6）。中线芽然后从内胚层分离，扩大双边在E13.5融合与气管两侧的鳃组织。最后，甲状腺组织到毛囊和C-细胞分化。 甲状腺形成目前的知识主要来自固定的组织的组织学分析，但涉及甲状腺形成的形态发生的事件是高度动态的，并涉及不同的细胞类型之间以及与细胞外基质的通信和交互。最近的工作表明，甲状腺细胞祖细胞产生高水平的VEGF招募内皮细胞向显影甲状腺，并且，反过来，招募了内皮细胞的促滤泡形成和C-细胞分化7。 大多数体外研究对甲状腺孤立成人甲状腺来源的细胞已被执行，无论是种植在二维组织培养塑料ðishes或3D矩阵。使用这些类型的文化，差异化的滤泡细胞要么保持极化和组织为毛囊或重新获取三维组织8-10。然而，这些纯上皮细胞，由它们的生理环境中取出的，并且在二维培养，忽略与细胞外基质，细胞因子，生长因子和其它细胞类型，如内皮细胞或神经细胞，它们在体内 ，通常遇到的相互作用。一个很漂亮的研究最近描述了胚胎干细胞分化成的协议使用3D基质胶11的最终培养步骤甲状腺滤泡。然而，这些3D的文化缺乏与其他类型的细胞接触。 根据以往的专业知识对胰腺和唾液腺器官培养12-14，解剖小鼠胚胎甲状腺原基和培养上semiporous过滤器或显微镜塑料滑梯外植体的方法被开发了。 何时与E12.5胚胎工作时，甲状腺原基（中线原基和两个侧鳃体）的双重起源组织所施加的大片段的显微解剖。这包含在气管，但不是食道，并从咽弓的动脉到杓状肿胀延长。当培养于过滤器时，中线原基横向延伸的气管的每一侧，在那里它们与鳃体融合形成两个甲状腺瓣，通过一个狭窄的峡部仍然连接。 在文化方面，上皮细胞增生，组织成毛囊和分化为甲状腺细胞和C-细胞，这取决于它们的起源。包含在显微组织的内皮细胞也增殖和侵入甲状腺瓣到最后与上皮滤泡结构独立地血液流动或循环的因素，密切关联。为外植体的发展忠实地概括在体内发展换货，这种文化系统是最佳​​的学习形态和甲状腺发育过程中出现的分化事件。 甲状腺组织可以从野生型，基因敲除或荧光转基因胚胎获得，并且该培养系统是服从损耗和增益函数的实验。最后，在显微镜塑料菜荧光标记的甲状腺植时间推移成像可能被利用来更好地调查发生在体内的动力学和形态发生的动作的连续性。时间推移成像已被用于研究胰腺15,16或输尿管芽17的分支形态。

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">&#181;-slide 8 well, ibiTreat, tissue culture treated, sterile</td>
			<td style="width:85px;">proxylab</td>
			<td style="width:119px;">80826</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type I, rat tail</td>
			<td style="width:85px;">Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin from human plasma</td>
			<td style="width:85px;">Invitrogen</td>
			<td># 33010-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">M199</td>
			<td style="width:85px;">Invitrogen</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">HBSS</td>
			<td style="width:85px;">Invitrogen</td>
			<td>14025-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Tungsten wire</td>
			<td style="width:85px;">Goodfellow</td>
			<td style="width:119px;">LS237450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">culture plate insert (12 mm diameter)</td>
			<td style="width:85px;">Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">GlycoBlue</td>
			<td style="width:85px;">Invitrogen</td>
			<td style="width:119px;">AM9516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">E-cadherin</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">610182</td>
			<td>1/1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Ezrin</td>
			<td style="width:85px;">Thermo Scientific</td>
			<td style="width:119px;">MS-661-P1</td>
			<td>1/400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">PECAM</td>
			<td style="width:85px;">BD Biosciences</td>
			<td style="width:119px;">550274</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Hoechst</td>
			<td style="width:85px;">Sigma</td>
			<td style="width:119px;">B2261</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo culture system to study thyroid development

甲状腺是一个bilobated内分泌腺定位在颈部的基部，产生甲状腺激素T3，T4，和降钙素。 T3和T4是由区别的甲状腺细胞，组织在封闭的球体称为卵泡产生的，而降钙素是由C-细胞，穿插在卵泡和密集的网络毛细血管之间的合成。虽然成人甲状腺结构和功能已被广泛地描述和研究，对“血管滤泡”单位，C-细胞在软组织的分布和上皮细胞和内皮细胞之间的旁分泌通信的形成远未了解。 这种方法描述了semiporous过滤器或显微镜塑料滑梯的连续步骤的小鼠胚胎甲状腺原基剥离和它的文化。在为期四天，这个文化系统忠实地概括体内甲状腺发展。事实上，（ⅰ）亿的脏器obation发生时（E12.5植），（ⅱ）前体甲状腺组织成毛囊和极化，（ⅲ）甲状腺细胞和C-细胞分化，以及（iv）中存在的显微组织增殖的内皮细胞，迁移到甲状腺叶，并紧密地与上皮细胞联系起来，因为他们在体内 。 甲状腺组织可以从野生型，基因敲除或荧光转基因胚胎获得。此外，外植体培养可以通过添加抑制剂被操纵，阻断抗体，生长因子，或什至细胞或条件培养基。 体外发育可以实时进行分析，或在培养的任何时间，通过免疫染色和RT-qPCR的。 总之，甲状腺外植体培养结合下游全贴或部分成像和基因表达分析提供了强大的系统，用于操纵和研究甲状腺邻形态发生和分化活动rganogenesis。

甲状腺原基（中线和UB连同周围组织）和甲状腺瓣从小鼠胚胎在E12.5和e13.5/e14.5分别（ 图1）解剖。一天培养上过滤后，中线原基是可见的（ 图2A），为一细长的组织生成树上的气管的上方。它逐渐形成于气管的每一侧两个波瓣。如果孤立甲状腺裂片（E13.5或E14.5）进行培养时，它们会扩展，经受形态发生和上皮细胞将组织成肉眼可见滤泡结构（ 图2B）。 <p cl...

Watch this Scientific Journal Video about An Ex vivo Culture System to Study Thyroid Development at JoVE.com

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 ...

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An Ex vivo Culture System to Study Thyroid Development

The Endocrine System

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11.5K 次观看.  Université catholique de Louvain & de Duve Institute. 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.

视频: An Ex vivo Culture System to Study Thyroid Development

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 ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

封闭的系统使用透氧材料科技HYPERStack船只的细胞培养协议

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

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 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 ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Methods Article

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