这项工作得到了纽约国家办事处的支持, 为发展中残疾人士和胎盘分析有限责任公司, 新的拉罗谢尔, 纽约。

本议定书遵循了纽约国家发展残疾基础研究研究所人类研究伦理学委员会的指导方针。
1. 长柔毛树解剖
<ol><li>用磷酸盐抛光生理盐水 (PBS)14冲洗福尔马林固定胎盘组织, 在解剖显微镜的舞台上取出福尔马林并放置在培养皿中。使用手术刀和细钳, 以逗胎盘组织分开寻找长柔毛树 (白色丝状分支结构)。解剖出绒毛树 (几毫米长) 的碎片, 将组织转移到含有1毫升 PBS 的微管上。</li>
</ol>2. 免疫荧光染色
<ol><li>用吸管, 取出 pbs, 换成新鲜的 pbs。让我们站10分钟, 偶尔旋转。再重复一次。 注意: 这一步和所有进一步的步骤都是在室温下进行的, 除了主要的抗体孵化。</li>
	<li>使用吸管, 去除和取代 pbs +0.1% Triton-X100 + 2% 山羊血清 (pbs t GS) permeabilize 组织和阻断非特异性的二级抗体结合。孵育30分钟。</li>
	<li>用2% 山羊血清、小鼠单克隆 anti-CK7 和兔 anti-CK7 的 pbs 取代 pbs T GS。<ol>
<li>孵化组织过夜在4摄氏度。</li>
		<li>清除其余的抗体, 用 PBS-GS 清洗10分钟, 偶尔旋转。</li>
		<li>移除 pbs gs 并替换为包含山羊抗小鼠 igg 的 pbs gs, 再加上一个绿色发射 (520 nm) 荧光和山羊抗兔 igg 结合红外发射 (652 nm) 荧光。</li>
		<li>重复步骤2.1 删除抗体。</li>
		<li>将 immunolabeled 组织在室温下存放在黑暗中。</li>
	</ol>
</li>
</ol>3. 组织的澄清
<ol><li>用 PBS 在10分钟间隔内冲洗样品3次。</li>
	<li>通过用吸管去除 PBS 并用50% 乙醇取代它 (甲醇也可用) 来脱水组织。孵育10分钟, 用新鲜的50% 乙醇代替, 孵育10分钟. 再重复一次, 总共有3孵化。重复孵化与70%。然后用100% 乙醇代替, 孵育15分钟 (或者甲醇可以使用)。</li>
	<li>用一个细尖钳, 转移组织到一个新鲜的微管含有〜1毫升 Solution-1 4 小时。</li>
	<li>用细尖钳, 将组织转移到含有 Solution-2 的新鲜微管中4小时。 注意: 当储存在黑暗中时, 免疫荧光在室温下稳定至少6月。</li>
	<li>不要与任何水溶液混合, (例如安装介质), 因为清除将丢失。</li>
</ol>4. 显微镜安装
<ol><li>不要在标准的玻璃滑梯和盖玻片之间安装组织, 因为组织是软的, 容易变形。</li>
	<li>请参阅图 5B并在赛克斯摩尔会议厅中安装该组织, 如下所示;<ol>
<li>用细尖钳放置一个25毫米圆盖玻片在房间基地。</li>
		<li>用细尖钳把橡胶垫片放在底座上的盖玻片上。</li>
		<li>用吸管放置一滴 (~ 40 µL) 的 Solution-2 在盖玻片的中心。</li>
		<li>用细尖钳将一块组织从 Solution-2 转移到盖玻片中心的下落。</li>
		<li>在垫片顶部放置第二盖玻片。</li>
		<li>将锁定环放入底座顶部, 压缩盖玻片和垫片直至牢固。 注意: 小心不要过度收紧或盖玻片会粉碎。</li>
		<li>将22口径的针插入底座和垫片的端口。</li>
		<li>用1.5 毫升的 Solution-2 填充3毫升的注射器。</li>
		<li>在注射器上安装一个25口径的针头。</li>
		<li>将注射器针插入到相反的端口并通过垫片。</li>
		<li>确保室内空气通过其他25口径针, 轻轻注入 solution-2and 填充腔内。</li>
		<li>取出针头和注射器, 将房间放在共焦显微镜舞台上的大玻璃滑梯上。</li>
	</ol>
</li>
	<li>或者, 从硅烷板上切割自定义井。<ol>
<li>用剪刀切割 25.4 x 25.4 毫米2件从该板。</li>
		<li>用手术刀切开 ~ 12.7 x 12.7 毫米2在片断。</li>
		<li>把一块牢牢地压在显微镜滑梯上。</li>
		<li>用 Solution-2 填满井顶。</li>
		<li>将组织片从微管转移到井中心。</li>
		<li>用细尖的镊子把盖玻片放在井顶, 这样盖玻片的底部就会被 Solution-2 浸湿。</li>
		<li>在共焦显微镜阶段安装与组织的克斯克摩尔室组件。</li>
	</ol>
</li>
</ol>5. 共焦显微术
<ol><li>将细胞中的组织集中在10x 的目标上。</li>
	<li>在焦距平面上上下移动显微镜, 测量组织的顶部和底部之间的距离。</li>
	<li>移动舞台在2.5 µm 步骤收集一个共焦图像文件, 其中包含一组图像通过组织从上到下。</li>
</ol>6. 逆转清算
<ol><li>通过将组织转移到含有 > 20x 的微管中以100% 乙醇的体积来逆转清除。在1小时间隔替换与70% 乙醇, 然后50% 乙醇和最后 PBS。存放在4摄氏度在黑暗中。 注: 现在处理组织的标准石蜡嵌入, 切片和组织学或免疫组织化学染色。</li>
</ol>7. 反褶积
注意: 对于以下步骤, 软件命令、选项卡和下拉菜单是斜体的。
<ol><li>启动反褶积软件并打开图像文件。</li>
	<li>输入 "夏天y" 窗口中列出的参数。<ol>
<li>对于 "间距" , 在µm 中输入体素 x、y 和 z 维度。</li>
		<li>对于每个颜色通道, 从下拉菜单中选择用于染色的荧光染料。</li>
		<li>对于 "情态", 请从下拉菜单中选择 "激光扫描共焦"。</li>
		<li>对于 "物镜", 请从下拉菜单中选择 "10x"。</li>
		<li>对于 "浸入介质", 从下拉菜单中选择 "空气"。</li>
		<li>单击 "应用"按钮。</li>
	</ol>
</li>
	<li>单击 "Deconvolution" 选项卡。</li>
	<li>在下拉菜单中单击 "3D 反褶积" 。</li>
	<li>在 "3D-卷积"窗口中, 确保设置为: 1) 自适应的 psf 盲、2) 理论的 psf、3) 使用 (默认自适应的 psf、10迭代、中等噪声)、4) 基名称。</li>
	<li>单击 "绿色复选按钮"启动程序。</li>
	<li>完成后, 重复步骤 7.3-7.6 以重新启动程序。当完成第三次和最后一次重新启动它。</li>
	<li>在 "数据管理器"窗口中, 单击以 "10-10-10-" 为前缀的文件。</li>
	<li>单击 "文件"选项卡, 然后单击 "另存为"。</li>
	<li>在 "保存s" 窗口中, 从 "数据类型"下拉菜单中选择 "8bit 无符号整数" , 然后单击 "保存"。</li>
</ol>8. 图像处理
<ol><li>启动斐济软件。<ol>
<li>单击 "文件", 单击 "打开"并选择 Deconvolved 图像文件, 然后单击 "打开" 窗口中的 "打开"按钮。</li>
		<li>单击 "图像/颜色/分割通道" , 然后将三色图像文件转换为仅包含一种颜色的三个新文件。保存包含 immunolabeled 长柔毛毛细管图像集的红色图像文件。删除绿色和蓝色图像文件。</li>
		<li>用以下方法减去红色图像文件中的背景荧光: "处理/减去背景"。</li>
		<li>在下拉菜单中, 将滚动球半径设置为10像素。</li>
		<li>保留未选中的4菜单选项, 然后单击 "确定"。</li>
		<li>单击 "进程堆栈" 菜单上的 "是" 。</li>
		<li>通过打开 "图像/调整/亮度对比度"菜单并调整最小、最大、亮度和对比度设置, 删除固有的组织荧光 (自发荧光)。注意不要去除任何毛细血管免疫荧光。</li>
		<li>通过打开 "图像/调整/阈值"菜单并调整滑块以突出显示毛细管免疫荧光, 以最小化剩余的无关荧光。检查 "深色背景"框, 然后单击 "应用"。在 "转换为二进制下拉"菜单上, 选中 "黑色背景"框, 然后单击 "确定"。保存黑白 (二进制) 图像文件以进行分析。</li>
	</ol>
</li>
</ol>9. 分析
<ol><li>对象计数和骨架。<ol>
<li>选择在步骤8.1.8 中创建的二进制图像文件。</li>
		<li>单击 "分析/3D OC 选项" , 然后在下拉菜单中只检查 "卷"和 "存储结果..."框。确保未选中"显示数字"框。单击 "确定" 按钮.
</li>
		<li>"Analyze/3D 对象计数器"上的 Click。在下拉菜单中, 将 "大小筛选: 最小值"设置为5000。确保未选中 "在边缘上排除对象"框。检查是否选中了 "对象的、统计信息和"摘要 "框。单击 "确定" 按钮。 注意: 这可能需要几分钟时间, 具体取决于文件大小和计算机配置。</li>
		<li>保存统计数据表和对象映射文件。</li>
		<li>选择对象映射文件。单击 "插件/骨架/缩略2维-3 d"以缩略二进制文件中的毛细管网络。单击 "文件/另存为"。在下拉菜单中选择 "Tiff......" 。在 "另存为 TIFF" 窗口中, 将 "Skel" 添加到对象映射文件名中, 然后单击 "保存"按钮.
</li>
		<li>单击 "分析/骨架/分析骨架 2 d-3 d"。在下拉菜单中接受默认设置, 然后单击 "确定" 按钮。保存分支"信息"表、"结果"表和两个输出文件 (Skel 对象标记为骨架和带标记的骨架)。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

机构审查委员会批准了收集胎盘绒毛组织选择性终止妊娠的福尔马林固定。在执行这些程序之前, 对医疗记录作了简要的回顾, 指出了孕产妇年龄、均等, 并确认没有任何潜在的医疗 (如例如、高血压、糖尿病和狼疮) 或胎儿 (结构或染色体异常,异常生长) 进行。数据表和任何收集的标本只用研究 ID 标记。因此, 所有标本都在离开操作套件之前被取消标识。绒毛由训练有素的观察者收集;去除了...

我们对发育中胎盘及其病理的理解, 在很大程度上局限于推断相邻绒毛之间的空间关系以及从组织学切片中提取的毛细血管。在这项研究中, 我们通过开发方法来生成三维 (3D) 的人胎盘毛细管网络的效果图, 这些结果适合于分析毛细管网络特征 (例如分支、固态)。为此, 我们将免疫荧光染色与两种商业组织清洁产品、Visikol-1 和 Visikol-2 (以下简称 Solution-1 和 Solution-2) 相结合。
人胎盘是一个巨大的复杂的血管位于母亲的 intervillous 血液和发育中的胎儿之间的接口。脐带从插入到绒毛膜板中延伸出来, 在动脉和静脉网络中 ramify, 用精心制作的血管网络覆盖绒毛膜表面。他们的末端然后击穿入胎盘盘的内部或深度, 在那里他们接受几个分支世代并且结束在末端绒毛和他们包含的毛细管网络, 气体交换的地点, 营养素和新陈代谢胎儿和母体血液之间的浪费。
对胎盘毛细管网络的侮辱在发展期间有持久的后果为胎儿、新生儿和紧急成人1,2,3的健康。鉴于妊娠相关的病症, 如流产, 宫内生长限制, 先兆子痫和孕妇糖尿病4,5,6有一个很高的价值, 制定测量方法和胎盘绒毛毛细管网络的表征。一个主要的障碍是胎盘血管网络包含广泛的范围。表面血管网络的直径可达4-5 毫米。末端具长柔毛毛细血管的顺序为 10-20 µm 直径;胎盘含有超过300公里的血管7。目前, 很少有易于使用和快速的技术, 可以捕捉这些极端的船只规模。到目前为止, 只有少量的绒毛可以通过显微镜来呈现。例如, Jirkovska et将重点放在足月胎盘绒毛上, 将共焦显微术与连续光学切片结合, 从120µm 厚剖面获得1µm 间隔;未提供所研究的样本数和统计数据, 8。对毛细管结构进行了鉴定, 并对绒毛和毛细血管的轮廓进行了手工绘制, 并将追查导出用于图像分析。虽然作者们讨论了他们的发现对 "生长的长柔毛血管网" 的影响, 但这样的结论是有问题的, 只有 "期限" (36 + 周孕龄) 组织的研究。类似地, 梅奥et al 和皮尔斯et等. 依赖于同一年龄的组织, 用于模拟血液流动和氧转运, 但其分析仅限于几个术语, 终端绒毛9,10.视学也被应用于研究绒毛状血管的结构。但同样的, 重点通常是以后怀孕分娩 liveborn 婴儿有一个或多个妊娠并发症11,12。
直到最近, 共焦显微术被限制为100-200 µm 的组织深度的成像, 因为吸收的激发和发射荧光的覆盖组织13 。虽然组织清除和3D 组织学已被广泛描述的文献和有许多方法, 组织清除许多不适合使用与组织一般, 因为他们不可逆转地破坏细胞形态学通过 hyperhydration蛋白质或脂肪的去除。因此, 无法验证这些结果是否表明了组织本身, 而不是加工工件, 而我们的组织清除过程是一种可逆的技术, 能够验证传统的组织学。组织清除一般涉及三种主要方法之一: 1) 在 RI 匹配解中, 对组织成分的折射率 (ri) 进行均匀匹配, 消除由于连续而引起的透镜的累积光散射。低 ri (细胞质) 和高 ri (蛋白质/脂) 成分的波动;2) 将脂质成分嵌入水凝胶中, 利用电泳/扩散法去除脂质成分;3) 蛋白质结构的扩张/变性, 以增加溶剂的穿透力, 以鼓励 RI 13的均匀性。虽然这些方法可以使组织透明, 并允许生成生物标志物的3D 表示, 但这些3D 表示有疑问的临床价值, 因为它是挑战, 以确定这些图像是否预示着组织的性质或组织清除过程。另一方面, 由于我们的组织清除是可逆的标准组织学和/或免疫组化可以应用到同一组织, 以评估是否有临床意义的改变。
本研究对47例从23例正常和选择性终止妊娠到9-23 个完成周妊娠期和两次足月正常分娩的远端长柔毛标本进行了分析。免疫荧光标记滋养层和内皮已经使对长柔毛血管网络的变化及其复杂性进行了定量和自动化的分析。
通过该协议, 我们对终端绒毛及其毛细管网络进行了前所未有的分离和分析。这种方法, 当应用到长柔毛和毛细管网络发展横跨妊娠将确定这些属性是出生的健康儿童的基础。当应用于复杂妊娠的研究时, 它还将阐明胎盘病变何时以及如何改变其鞘的绒毛状树木和毛细血管网络, 以及这些如何影响胎儿的福祉。

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			<td height="20" style="height:20px;width:435px;">10% Formaldehyde solution (w/v) in aqueous phosphate buffer</td>
			<td style="width:199px;">Macron Fine Chemicals</td>
			<td style="width:127px;">H-121-08</td>
			<td style="width:517px;">General fixation agent, ready to use formula, use caution as vapors are toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blades</td>
			<td style="width:199px;">ThermoFisher Scientific</td>
			<td style="width:127px;">08-916-5B No. 11</td>
			<td style="width:517px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel</td>
			<td style="width:199px;">ThermoFisher Scientific</td>
			<td style="width:127px;">08-913-5</td>
			<td style="width:517px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine forceps</td>
			<td style="width:199px;">Electron Microscopy Services</td>
			<td style="width:127px;">78354-119</td>
			<td style="width:517px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Micro Tube (1.7 mL)</td>
			<td style="width:199px;">PGC Scientifics</td>
			<td style="width:127px;">505-201</td>
			<td style="width:517px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:127px;">D8537</td>
			<td style="width:517px;">PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette</td>
			<td style="width:199px;">VWR</td>
			<td style="width:127px;">52947-948</td>
			<td style="width:517px;">disposable, 3ml transfer pipette</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td style="width:199px;">Boehriner Mannheim</td>
			<td style="width:127px;">789 704</td>
			<td style="width:517px;">Dilute to 0.1% from stock</td>
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			<td height="21" style="height:21px;">Goat Serum</td>
			<td style="width:199px;">Gibco</td>
			<td style="width:127px;">16210-064</td>
			<td style="width:517px;">Dilute to 2% in PBS solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse monoclonial anti-ck7 Keratin 7 Ab-2 (Clone OV-TL 12/30)</td>
			<td style="width:199px;">ThermoFisher Scientific</td>
			<td style="width:127px;">MS-1352-RQ</td>
			<td style="width:517px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit Anti-CD31 antibody</td>
			<td style="width:199px;">Abcam</td>
			<td style="width:127px;">ab28364</td>
			<td style="width:517px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">green emitting (520 nm) fluorochrome&#160;</td>
			<td style="width:199px;">Invitrogen</td>
			<td style="width:127px;">A11017</td>
			<td style="width:517px;">Alexa-Fluor 488</td>
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		<tr height="21">
			<td height="21" style="height:21px;">infrared emitting (652 nm) fluorochrome</td>
			<td style="width:199px;">Invitrogen</td>
			<td style="width:127px;">A21072</td>
			<td style="width:517px;">Alexa Fluor 633</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Ethanol alcohol 200 proof</td>
			<td style="width:199px;">Pharmco-Aaper</td>
			<td style="width:127px;">111000200</td>
			<td style="width:517px;">Dilute down to lower concentrations using PBS as needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solution-1</td>
			<td style="width:199px;">Visikol Inc.</td>
			<td style="width:127px;"></td>
			<td style="width:517px;">Visikol Histo-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solution-2</td>
			<td style="width:199px;">Visikol Inc.</td>
			<td style="width:127px;"></td>
			<td style="width:517px;">Visikol Histo-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skyes-Moore chambers</td>
			<td style="width:199px;">BellCo Glass Inc.</td>
			<td style="width:127px;">P/N 1943-11111</td>
			<td style="width:517px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">25 gauge needle</td>
			<td style="width:199px;">ThermoFisher Scientific</td>
			<td style="width:127px;">14-826AA</td>
			<td style="width:517px;">BD Precision Glide Needes</td>
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		<tr height="21">
			<td height="21" style="height:21px;">3 mL syringe</td>
			<td style="width:199px;">ThermoFisher Scientific</td>
			<td style="width:127px;">14-823-40</td>
			<td style="width:517px;">BD disposable syringe</td>
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			<td height="21" style="height:21px;">PDMS silicon sheets</td>
			<td style="width:199px;">McMaster-Carr</td>
			<td style="width:127px;">P/N 578T31</td>
			<td style="width:517px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">confocal microscope</td>
			<td style="width:199px;">Nikon Inc.</td>
			<td style="width:127px;"></td>
			<td style="width:517px;">Nikon C1 Confocal Microscope</td>
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			<td height="21" style="height:21px;">Deconvolution software</td>
			<td style="width:199px;">Media Cybernetics</td>
			<td style="width:127px;"></td>
			<td style="width:517px;">AutoQuant X22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiji image processing software</td>
			<td style="width:199px;"></td>
			<td style="width:127px;"></td>
			<td style="width:517px;">free, Open source&#160; software available at https://fiji.sc</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin</td>
			<td style="width:199px;">Leica Biosystems</td>
			<td style="width:127px;">3801570</td>
			<td style="width:517px;">Component 1 of SelecTech H&#38;E staining system</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Alcoholic Eosin</td>
			<td style="width:199px;">Leica Biosystems</td>
			<td style="width:127px;">3801615</td>
			<td style="width:517px;">Component 2 of SelecTech H&#38;E staining system</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Blue Buffer</td>
			<td style="width:199px;">Leica Biosystems</td>
			<td style="width:127px;">3802918</td>
			<td style="width:517px;">Component 3 of SelecTech H&#38;E staining system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aqua Define MCX</td>
			<td style="width:199px;">Leica Biosystems</td>
			<td style="width:127px;">3803598</td>
			<td style="width:517px;">Component 4 of SelecTech H&#38;E staining system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:435px;">Immunohistochemistry detection system</td>
			<td style="width:199px;">ThermoFisher Scientific</td>
			<td style="width:127px;">TL-125-QHD</td>
			<td>UltraVision Quanto Detection System HRP DAB</td>
		</tr>
	</tbody>
</table>

three-dimensional rendering and analysis of immunolabeled, clarified human placental villous vascular networks

母亲和胎儿之间的养分和气体交换发生在母体 intervillous 血的界面和巨大的长柔毛毛细血管网络中, 构成了人类胎盘的大部分实质。远端长柔毛毛细管网络是胎儿血液供应的终点后, 几代分支的血管延伸出脐带。这个网络有一个连续的细胞鞘, 合胞滋养屏障层, 防止胎儿血液和产妇血液的混合, 在其中持续沐浴。对胎盘毛细血管网络的完整性的侮辱, 发生在诸如孕产妇糖尿病、高血压和肥胖等疾病中, 会给胎儿、婴儿和成人带来严重的健康风险。为了更好地定义这些侮辱的结构效果, 为这项研究制定了一项协议, 它捕获毛细管网络结构的顺序为 1-2 毫米3 , 其中一个可以调查其拓扑特征的完全复杂性。为达到这一目的, 对胎盘端绒毛簇进行了解剖, 滋养层和毛细血管内皮 immunolabeled。然后用新的组织清理过程澄清这些样本, 从而可以获得共焦图像栈到 z 深度的 ~ 1 毫米。然后对这些堆栈的三维渲染进行处理和分析, 以生成基本的毛细管网络度量, 如体积、毛细管分支数和毛细管分支端点, 以验证这种方法是否适合毛细管网络特性。

从8周孕龄到足月分娩, 将人胎盘端绒毛簇中毛细血管的生成3D 效果计算为个体簇和骷髅进行网络分析。胎盘的功能单位 (图 1A) 是绒毛树, 是它们穿透胎盘实质的表面血管的延伸 (图 1B, 并在1C中放大)。然后 immunolabeled 解剖树的远端部分 (Figure1D和1E) 的碎片, 以显示绒?...

Watch this Scientific Journal Video about Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks at JoVE.com

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

本研究提出了一种可逆转组织清除、染色、3 d 渲染和分析人胎盘绒毛样品中血管网络的方法, 其顺序为 1-2 毫米3。

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

three-dimensional-rendering-analysis-immunolabeled-clarified-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.6K 次观看.  The New York State Office for People with Developmental Disabilities. 本研究提出了一种可逆转组织清除、染色、3 d 渲染和分析人胎盘绒毛样品中血管网络的方法, 其顺序为 1-2 毫米3。

视频: Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

科研

发育生物学

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

牙龈三维炎症人体组织当量

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

高频超声在胎儿和胎盘发育分析中的影响

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

从多能干细胞中无血清自由生产三维人类肝球

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...