本研究得到了浙江省自然科学基金资助（Z23H290001，LY19H280001）;国家自然科学基金（82274364、81673607、81774011）;以及湖州市科技基金公益研究项目（2021GY49,2018GZ24）。感谢浙江中医药大学中医科学院医学研究中心公共平台的大力帮助、技术支持和实验支持。

1. 材料准备
<ol><li>在12mL的DMEM培养基（10%胎牛血清和100U / mL青霉素 - 链霉素）中培养2 x 106 Panc02细胞，并在5%二氧化碳和95%空气的气氛中保持在37°C和95%湿度48小时。</li>
	<li>收集Panc02细胞，以28× g 离心2分钟，然后弃去上清液。确保样品具有适当的大小（1 x 107 个细胞），否则，以下固定和脱水步骤将无法正常工作。</li>
	<li>加入1mL的2.5%戊二醛作为固定剂，将新鲜的Panc02细胞固定在4°C的1.5mL微量离心管中过夜。 注意:在以下每个步骤（步骤1.3-1.11）中，样品需要在1,006× g 下离心5分钟。</li>
	<li>吸出固定剂，并用0.1M磷酸盐缓冲盐水（PBS）冲洗样品两次，然后在室温下用双蒸水（ddH2O）冲洗两次，每次10分钟。</li>
	<li>加入50μL含有1%四氧化锇（OsO 4）和1.5%亚铁氰化钾的溶液，比例为1:1，在4°C下1小时。然后，用0.1M PBS冲洗两次，每次10分钟，并用ddH2O冲洗10分钟。</li>
	<li>加入 1 mL 的 1% 硫代碳酰肼 （TCH），在室温下孵育 30 分钟至 1 小时，然后用 ddH2O 冲洗四次，每次 10 分钟。</li>
	<li>加入 50 μL 的 1% OsO4，在室温下固定 1 小时，然后用 ddH2O 冲洗四次，每次 10 分钟。</li>
	<li>在4°C下加入1mL的2%乙酸铀酰过夜，然后用ddH2O冲洗四次，每次10分钟。</li>
	<li>在60°C下加入1mL沃尔顿溶液（0.066g硝酸铅溶解在10mL的0.03mol/L天冬氨酸储备溶液中），再孵育1小时。</li>
	<li>用ddH2O冲洗四次，每次10分钟，然后在分级酒精系列中脱水10分钟，每次:50%酒精，70%酒精，80%酒精和90%酒精1x和100%酒精2x。然后，在100%丙酮中脱水两次，每次10分钟。使用每种溶液 1 mL 进行脱水。</li>
	<li>将 200 μL 丙酮与 Pon 812 环氧树脂以 3:1、1:1 和 1:3 的比例混合。将样品分别在室温下浸泡在树脂中2h，4h和4h，然后取100%Pon 812环氧树脂浸渍过夜。 注意:执行染色和树脂包埋步骤时，重要的是在通风橱中操作，因为这些是有毒物质。此外，在实验过程中必须穿上实验室外套和耐溶剂手套。</li>
	<li>将试样放入包埋模具中，然后置于60°C的烘箱中48小时聚合。使用平面包埋29 来减少样品周围树脂的外观。这简化了试样安装，并减少了充电伪影对后续图像分割的影响。 注意: 为了随后生成水平切割表面，可以将少量树脂添加到模具孔中，注意不要过度填充。</li>
	<li>首先清洗硅片，然后用浓缩的H2SO4 / H 2 O2溶液亲水30分钟来制备硅片。使用超薄切片机在亲水硅晶片上收集厚度为70nm的连续切片条，并在60°C烘箱中干燥10分钟。 注意:当切片漂浮到晶圆表面时，请缓慢捕获切片，以防止切片丢失或变形。</li>
</ol>2. 图像采集与三维重建
<ol><li>在将硅晶圆安装到载物台上之前，用蒸馏水清洗切片三次，然后风干。切割尺寸为1 cm x 0.5 cm的导电碳带，并将其粘贴在硅晶片和SEM样品台之间以完成样品设置（图2E）。</li>
	<li>将 FE-SEM 加速电压参数设置为 2 kV，工作距离为 4 mm（图 3B 和 表 1）。 注意:为了提高信噪比并减少图像中的噪声，请尽可能以低放大倍率进行观察。随着倍数的增加，SEM的扫描范围减小，导致图像上的电荷积累增加。</li>
	<li>单击顶部菜单栏中的 H/L 图标 。首先，在低放大倍率下，定向目标切片条的第一部分，然后再次单击 H / L 选项（图3A）切换到高放大倍率模式;通过调整图像亮度、对比度和放大率，为感兴趣的结构收集适当的图像。</li>
	<li>选择" 项目视图">"开放数据"，然后将要分析的图像文件导入软件。</li>
	<li>通过单击对齐 切片>编辑 对齐图片（图 4B），并通过修改图像透明度来调整左下角菜单栏中的 强度范围 值。在这里，使用半自动对齐策略;具体来说，通过自动对齐，使图片与上一张图片重叠，然后进行手动微调。 注意:在这项工作中，在对齐过程中，使用先前的图像作为参考，并利用移动和旋转操作来最大程度地重叠两个图像的目标结构。单击对齐 当前切片对 可以帮助自动对齐图像，但可能会出现错位。</li>
	<li>选择 "提取子体积"模块，并裁剪对齐的数据集以适合整个堆栈重叠部分的大小。</li>
	<li>在 分割 子部分（图 4C）下，选择重新 采样>分割>保存。选择 魔棒 的阈值和 画笔 工具的大小以选择正确的范围。这里再次采用半自动分割方法，首先使用 魔杖 自动选择合适的大面积区域，然后使用 画笔 准确描述细节。 注意:通过更改遮罩值并使用绘制极限线，可以根据感兴趣结构与周围结构之间存在的像素强度的明显差异，将魔棒应用于分割图像。它无法区分图像采集过程中产生的充电伪影。因此，它可能会导致目标区域的分段不正确。画笔工具可用于手动分割以准确重建生物结构。</li>
	<li>在 "分割>选择"下，单击 + 图标以添加所选区域（图 4C）。</li>
	<li>重复上述步骤（2.8）以在不同的图像中选择相同的目标结构，直到选择完成以重建感兴趣的微观结构。 注意:在线粒体重塑过程中，必须从一组连续图像中间的图片中执行目标对象的选择。如果从第一张或最后一张图片中选择所需区域，则重建结果将无法呈现完整的 3D 可视化。</li>
	<li>完成区域分割后，根据对象大小和图片分辨率的最佳结果生成图像文件。单击 裁剪编辑器，然后在 虚拟滑块 框中输入数字 6（图 4C）。</li>
	<li>在左侧的下拉菜单中，右键单击" 项目 "子部分下的灰色区域，然后选择" 生成曲面>创建>应用"。在生成的文件中，使用曲面视图模块创建 曲面 结构和 3D 表示。</li>
</ol>3. 量化
<ol><li>单击删除 小斑点>应用 （图 4D）。在 "项目 "子部分下，右键单击灰色区域，然后选择 "标签分析">"应用 "（图 4D）。</li>
	<li>自定义列的名称，然后选择度量值组。从"本机测量"框中选择"Volume3d 和 Length3d"。 </li>
	<li>单击屏幕左下角的 "应用 "按钮。复制数据，并在统计软件（如GraphPad）中绘制图形。</li>
</ol>

3D 技术可视化胰腺癌细胞中的线粒体嵴

作者声明不存在利益冲突。

这里介绍的方法是一个有用的分步指南，用于应用3D重建技术，该技术涉及将电子显微镜和图像处理技术应用于从连续超薄切片生成的2D断层扫描图像的堆叠和分割。该协议强调了可以通过细胞器超微结构的3D可视化来解决的2D图像的局限性，该图像具有高分辨率水平上结构的强可重复性和更高的精度的优点。更重要的是，这种3D可视化可以应用于肿瘤细胞，使病理机制的研究更加直接可靠。在这项...

线粒体是细胞中最重要的细胞器之一。它们是细胞生物能量学和代谢的中心枢纽1,2，并在癌症中起着关键作用3。胰腺癌（PC）由于其快速扩散和高死亡率，是最难治疗的癌症之一4。线粒体功能障碍主要由线粒体形态变化3,5,6,7引起，与PC8的疾病机制有关。线粒体也是高度动态的，这反映在其网络连接和嵴结构的频繁和动态变化上9。嵴结构的重塑可直接影响线粒体功能和细胞状态10,11，在肿瘤细胞生长、转移和肿瘤微环境变化过程中发生显著改变12,13。
近年来，科学家们使用EM观察研究了这种细胞器14,15,16,17;例如，研究人员使用3D重建技术分析了线粒体动力学6,7,18,19。电子显微镜图像三维重建的一般概念和方法早在196820年就正式确立，涉及结合电子显微镜、电子衍射和计算机图像处理来重建T4噬菌体尾巴。到目前为止，电子显微镜3D成像技术在图像分辨率21、自动化程度22和处理体积23方面取得了重大进展，并且在生物学研究中的应用越来越广泛，从组织水平到纳米尺度的细胞器超微结构水平24。近年来，电子显微镜3D成像也成为一种前景广阔的技术25,26,27。
对线粒体嵴的日益关注特别说明了超微结构体积成像的基本要求。透射电子显微镜（TEM）已被用于可视化在铜网格（400目）28上收集的样品，电子束穿过该切片。然而，由于铜网格的范围有限，不可能对同一样品29的连续切片进行完全成像。这使得TEM成像过程中靶标结构的研究变得复杂。此外，TEM依赖于耗时且容易出错的手动任务，包括切割和收集多个切片并按顺序成像21，因此它不适用于大体积样品的超微结构重建23。目前，大体积样品成像的高分辨率重建是通过使用专用设备实现的，例如TEM相机阵列（TEMCA）30 或两个第二代TEMCA系统（TEMCA2）31，可以在短时间内实现自动化高通量成像。然而，由于需要定制设备，这种类型的成像不具有易于获得和通用的优点。
与TEM相比，基于SEM 32,33自动生成大面积连续体积图像的方法提高了串行成像的效率和可靠性，并提供了更高的z分辨率34。例如，连续块面扫描电子显微镜（SBF-SEM）和聚焦离子束扫描电子显微镜（FIB-SEM）都使高速，高效和分辨率35,36的超微结构的3D重建成为可能。然而，不可避免地，通过SBF-SEM的金刚石刀或通过FIB-SEM33,37的聚焦离子束进行铣削，块表面被机械剃掉。由于两种方法对样品的破坏性，不可能再次重建相同的目标结构以进行进一步分析38,39,40。此外，很少有研究试图使用EM重建癌细胞的3D细胞器超微结构来观察病理变化12。基于这些原因，为了进一步阐明胰腺癌细胞等癌细胞的病理机制，我们提出了一种使用超薄切片机和场发射扫描电子显微镜（FE-SEM）在嵴水平分析线粒体超微结构的连续切片图像的3D重建新技术;通过这项技术，可以使用高效且可访问的方法获取高分辨率数据。使用超薄切片机制成的系列超薄切片可以半永久地存储在网格盒中，并在几年后多次重新成像41.FE-SEM因其能够提供高分辨率成像，高放大倍率和多功能性而作为各种研究领域的工具受到高度重视42。为了尝试以3D形式显示细胞器的精细结构，使用FE-SEM 43,44产生的背散射电子生成具有有用分辨率的串行2D图像堆栈的技术也可用于实现目标区域或其相关结构的高通量和多尺度成像，而无需特殊设备45.电荷伪影的产生直接影响采集图像的质量，因此保持较短的停留时间尤为重要。
因此，本研究详细阐述了该SEM技术中用于重建线粒体嵴46的3D结构的实验程序。具体来说，我们展示了使用Amira软件实现线粒体区域的半自动分割和数字化3D重建的过程，其中还包括使用传统的OTO标本制备方法44,47制作切片样品，使用超切片机切片完成切片收集，并通过FE-SEM获取顺序2D数据。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(1S,3R)-RSL3</td>
			<td>MCE</td>
			<td>HY-100218A</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>SIGMA</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>Amira</td>
			<td>Visage Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspartic acid</td>
			<td>MCE</td>
			<td>HY-42068</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#8217;s medium</td>
			<td>Gibco</td>
			<td>11995115</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrostatin-1</td>
			<td>MCE</td>
			<td>HY-100579</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10437010</td>
			<td></td>
		</tr>
		<tr>
			<td>Field emission scanning electron microscope</td>
			<td>HITACHI</td>
			<td>SU8010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Alfa Aesar</td>
			<td>A10500.22</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead nitrate</td>
			<td>SANTA CRUZ</td>
			<td>sc-211724</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide</td>
			<td>SANTA CRUZ</td>
			<td>sc-206008B</td>
			<td></td>
		</tr>
		<tr>
			<td>Panc02</td>
			<td>European Collection of Authenticated Cell Cultures&#160;</td>
			<td>98102213</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Biosharp</td>
			<td>BL505A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Biosharp</td>
			<td>BL302A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pon 812 Epoxy resin</td>
			<td>SPI CHEM</td>
			<td>GS02660</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide</td>
			<td>Macklin</td>
			<td>P816416</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide</td>
			<td>Merck</td>
			<td>223220</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>LEICA</td>
			<td>EMUC7</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>RHAWN</td>
			<td>R032929</td>
			<td>2020.2</td>
		</tr>
	</tbody>
</table>

a three-dimensional technique for the visualization of mitochondrial ultrastructural changes in pancreatic cancer cells

了解细胞器超微结构的动态特征，不仅具有丰富的未知信息，而且从三维（3D）角度来看也很复杂，对于机制研究至关重要。电子显微镜（EM）具有良好的成像深度，并允许重建高分辨率图像堆栈，以研究细胞器的超微结构形态，即使在纳米尺度上也是如此;因此，3D重建因其无与伦比的优势而变得越来越重要。扫描电子显微镜（SEM）提供了一种高通量图像采集技术，允许在连续切片中从相同的感兴趣区域重建3D大型结构。因此，SEM在大规模三维重建中的应用，以恢复细胞器的真实三维超微结构变得越来越普遍。在该协议中，我们建议结合连续超薄切片和3D重建技术来研究胰腺癌细胞中的线粒体嵴。该协议以逐步的方式描述了如何执行这些技术的详细信息，包括锇 - 硫代碳酰肼 - 锇（OTO）方法，连续超薄切片成像和可视化显示。

在细胞培养过程中（图1A），我们首先将胰腺癌细胞分为用完整培养基培养的对照组，（1S，3R）-RSL348（RSL3，铁死亡激活剂，100nM）组和RSL3（100nM）加铁抑素-149（Fer-1，铁死亡抑制剂，100nM）组。通过上述实验步骤，扫描电镜分别获取对照组、RSL3组和抑制剂组（RSL3+Fer-1组）的38张（补充图1）、43张（补充图2）和44?...

Watch this Scientific Journal Video about A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells at JoVE.com

作者聚焦:胰腺癌细胞线粒体超微结构变化可视化的三维技术  

该协议描述了如何重建线粒体嵴以实现高精度，高分辨率和高通量的3D成像。

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a ...

本项目旨在通过在亚细胞纳米尺度上改变结构，建立细胞器形态与癌症病理机制之间的联系，来评价药物的抗癌作用。该技术将传统的电子显微镜技术与三维重建技术相结合，无需专用设备。与其他技术相比，它可以提高成像效率和重建灵活性，并随时对目标结构进行现实生活中的重建。因此，它非常简单且通用。这种连续超薄载玻片的三维重建技术可以反复还原真实的立体化学，而无需特定的昂贵设备。它可以随时用于所有细胞器的定性和定量分析。实现了观察的简便操作。

a-three-dimensional-technique-for-visualization-mitochondrial

Research

JoVE Journal

Cancer Research

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

1.5K 次观看.  School of Pharmaceutical Science of Zhejiang Chinese Medical University. 本项目旨在通过在亚细胞纳米尺度上改变结构，建立细胞器形态与癌症病理机制之间的联系，来评价药物的抗癌作用。该技术将传统的电子显微镜技术与三维重建技术相结合，无需专用设备。与其他技术相比，它可以提高成像效率和重建灵活性，并随时对目标结构进行现实生活中的重建。因此，它非常简单且通用。这种连续超薄载玻片的三维重建技术可以反复还原真实?...

视频: 作者聚焦:胰腺癌细胞线粒体超微结构变化可视化的三维技术  

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

科研

癌症研究

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

研究结肠癌干细胞的球体三维模型

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Robotic Pancreatoduodenectomy for Pancreatic Head Cancer: a Case Report of a Standardized Technique

Robotic pancreatoduodenectomy (RPD) for pancreatic cancer is a challenging procedure. Aberrant vasculature may increase the technical difficulty. Several studies have described the safety of RPD in case of a replaced or aberrant right hepatic artery, but detailed video descriptions of the approach are lacking. This case report describes a step-&#173;by-&#173;step technical video in case of a replaced right hepatic artery. A 58-year-old woman presented with an incidental finding of a 1.7 cm pancreatic head mass. RPD was performed using the da Vinci Xi system and involves a robotic-assisted pancreatico- and hepatico-jejunostomy and open gastro-jejunostomy at the specimen extraction site. The operation time was 410 min with 220 mL of blood loss. The patient had an uncomplicated postoperative course and was discharged after 5 days. Pathology revealed a pancreatic head cancer. RPD is a feasible and safe procedure in case of a replaced hepatic artery when performed in selected patients in high-volume centers by experienced surgeons.

Robotic pancreatoduodenctomy (RPD) has been highly standardized in recent years and may be used in selected patients with pancreatic head cancer, including those with a replaced right hepatic artery. This case report describes a standardized and reproducible technique for RPD, which includes the approach of the Dutch LAELAPS-3 training program to an aberrant vasculature.

胰腺头癌机器人胰十二指肠切除术:标准化技术的病例报告

Robotic pancreatoduodenectomy (RPD) for pancreatic cancer is a challenging procedure. Aberrant vasculature may increase the technical difficulty. Several ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

磷脂介质诱导的三维培养物转化

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary gland secretion, circadian rhythm, macular degeneration, immune function, and cardiac physiology. Murine surgical sympathectomy is a method for studying the effects of adrenergic innervation by allowing for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects. However, surgical sympathectomy in mice is technically challenging because of the small size of the superior cervical ganglion. This study describes a surgical technique for reliably identifying and resecting the superior cervical ganglion to ablate the sympathetic nervous system. The successful identification and removal of the ganglion are validated by imaging the fluorescent sympathetic ganglia using a transgenic mouse, identifying post-resection Horner's syndrome, staining for adrenergic markers in the resected ganglia, and observing diminished adrenergic immunofluorescence in the target organs following sympathectomy. This model enables future studies of cancer progression as well as other physiological processes regulated by the sympathetic nervous system.

The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion.

小鼠模型中上颈神经节切除术的手术技术

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary ...