为这项工作提供资金是通过美国心脏协会 (16SDG29870005) 和北卡罗莱纳州大学研究和创新种子资金提供的。

1.一天 1: 细胞和试剂制备 注: 所有单元格和试剂的准备工作应在生物安全柜，防止样品污染。
<ol><li>温暖过滤杜尔贝科 &#39; s 改良鹰 &#39; s 介质 (DMEM)，胎牛血清 (FBS)，L-谷氨酰胺，并在 37 ℃ 水浴中青霉素/链霉素。</li>
	<li>准备新生儿人真皮成纤维细胞 (HDFn) 媒体通过补充温暖 DMEM 10%胎牛血清、 1%青霉素/链霉素和 1 %l-谷氨酰胺。</li>
	<li>解冻一瓶的冷冻 HDFns 和离心机在 200 x g 8 分钟删除超低温保存介质。悬在准备 HDFn 介质密度为 1 × 10 6 细胞/毫升和种子入 T-75 培养瓶中的细胞。</li>
	<li>地方烧瓶中孵化器 （37 ° C，5%CO 2），使细胞增殖。</li>
	<li>商店 HDFn 媒体在 4 ° C.</li>
	<li>根据需要准备 pro 和反 migratory 试剂。</li>
</ol> 2。第 3 天: 球体制备 <ol><li>执行以下操作在文化罩，以防止污染样品。</li>
	<li>当细胞到达 80%融合，吸出媒体和添加 5 毫升的 0.25%胰蛋白酶-EDTA。在 37 ° C 5 分钟要充分分离细胞从瓶表面孵化器存储瓶。</li>
	<li>将 5 毫升的温暖的媒体添加到瓶子以抵消胰蛋白酶。</li>
	<li>在微量离心管中，混合台盼蓝 10 μ L 和 10 μ L 细胞悬液。</li>
	<li>离心机在 200 x g.8 分钟将原始细胞悬浮液</li>
	<li>虽然被离心细胞悬液，台盼蓝细胞悬浮液 10 μ L 添加一个血球计数板和执行单元格计数。</li>
	<li>后离心，吸出媒体没有见面礼细胞团。1.25 x 10 5 细胞/毫升浓度时重悬细胞颗粒。</li>
	<li>10 厘米培养皿球体生长期间保持水分的环境的底部添加 5 毫升的无菌磷酸盐缓冲液 (PBS)。</li>
	<li>反转培养皿的盖子。添加几个 20 μ L 滴的培养皿盖的内部表面的细胞悬液。</li>
	<li>快速再次反转的盖子，并将它放回在底部的这道菜，小心不要取出挂滴。 图 1 说明了成功形成的挂滴。</li>
	<li>仔细将这道菜放在 37 ° C 的孵化器，并允许球体，成长在一个挂滴文化为期 72 h.</li>
</ol> 3。第 6 天: 嵌入球体三维矩阵 <ol><li>执行以下操作在文化罩，以防止污染样品。</li>
	<li>第一纤维蛋白层，形成创建足够的音量要加入 100 μ L 的凝块解决方案每井多井作为一个血块解决方案要求 （每井 1 球体）。血栓被由 10 %10 X HEPES 缓冲卷 （250 毫米复、 1500 毫米氯化钠、 50 毫米 CaCl 2、 pH 7.4）、 2 毫克/毫升纤维蛋白原和 0.1 U/mL 人 alpha 凝血酶。其余的凝块卷组成的超纯水。
	<ol><li>添加凝血酶最后和迅速将解决方案添加到所需井在 48 孔板以防止凝块聚合之前被添加到井。</li>
	</ol></li> <li>轻轻地摇/tap 孔板确保纤维蛋白凝块混合物很好，涂层整个，然后将板放在孵化器 1 h 允许底层凝块聚合。不动摇孔板难诱使气泡的形成;只是岩石板作为必要直至涂井的整体。如果使用较大的凝块卷，则这一步可能不必要。</li>
	<li>将球转移到纤维蛋白层。
	<ol><li>删除井板和培养皿中悬球体从孵化器除去文化并检查在显微镜下的球体。然后将盘子放入文化罩。</li>
		<li>仔细倒置培养皿允许访问的盖子给挂掉文化。</li>
		<li>使用附加到 1 毫升注射器，21 G 针小心转移一滴从倒盖成的每个井，中心照顾不来穿刺纤维蛋白聚合的层涂料的井底部。若要有效地传输下降，缓慢进针拉滴。别拉柱塞，尽快回来整个下落是内在的针;拉滴回来太远可能会引入空气气泡，可以破坏有效球体转让或随后成像。</li>
		<li>检查以确保球体有妥善转移到所有所需的油井在显微镜下孔板。</li>
	</ol></li> <li>第二纤维蛋白层的形成，创建另一个血块解决方案使用同一如上为筹备成立的第一次的纤维蛋白层 （步骤 3.2）。仔细移液器 100 μ L 至每个球体包含的下降与再次确保球体仍然完好无损，并已经不被推到了井的边缘在显微镜下研究。</li>
	<li>返回到孵化器孔板和允许第二个图层为 1 h.聚合</li>
</ol> 4。第 6 天: 加法的 Pro 洄游或抑制因子 <ol><li>温暖 HDFn 媒体到 37 ° C.</li>
	<li>执行以下步骤在罩，以防止污染。</li>
	<li>媒体准备包括适当浓度的 pro 反 migratory 代理要计算每个组。</li>
	<li>从孵化器和在文化罩的地方删除孔板。</li>
	<li>标准媒体每口井的控制球体组添加 500 μ L、 抑制介质对迁移抑制球体组，每个井 500 μ L 和 500 μ L 的 pro 洄游媒体每口井的迁移增强球体组。</li>
	<li>返回到孵化器的孔板。</li>
	<li>更改媒体每 48 h.</li>
</ol> 5。6-9 天: 评价细胞迁移和增殖 <ol><li>图像球体明视场显微镜使用另外的媒体 (0 h)，然后每隔 24 小时后立即 72 h.可选: 在每个时间点，以确定是否采取 z 堆栈图像迁移发生在飞机外的主要重点领域。在我们的经验这不会发生;然而，它是确保这是每个实验的情况非常有用。</li>
	<li>使用 ImageJ 来确定在每口井的细胞产物通过跟踪每个球体每个连续时间点的单元格边框和使用分析每个球体的面积的百分比增加 &#34; 措施 &#34; 函数获取地区。</li>
</ol>

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作者没有透露。

此协议允许细胞迁移在伤口愈合过程中检查关联 ECMs 通过体外的 3D 模型。过程的正确执行的关键一步是适当发展的成纤维细胞球体;制备过程中的细胞浓度进行了优化，给 2,500 细胞/除去、 初始浓度在 72 h 潜伏期可靠地允许球体到窗体。如果使用细胞数目不足，球体不可能有效地聚合，并且可能解体后被转移和嵌入到纤维蛋白层。如果使用不同的细胞类型，初始细胞浓度和 （或） 球体成熟...

伤口愈合是一个复杂的过程，从而导致组织完整性损伤后的修复。这个过程分为四个相互重叠的步骤，包括止血、 炎症、 扩散和重构，每个受规管的可溶性线索、 细胞与基质的相互作用和细胞间通讯的复杂组合。1,2这些线索业务流程控制大量的伤口愈合过程，重要的，包括细胞迁移中的细胞反应。3,4细胞迁移是一个高度动态的过程依赖于细胞表型和细胞外基质环境的生物化学和生物物理属性。5细胞不断探讨其细胞外基质环境通过整合素介导的途径;这一过程可使细胞感觉种类繁多的矩阵属性包括地形、 僵硬和约束。细胞迁移的二维 (2D) 分析演示迁移由板状伪足形成在前缘通过代肌动蛋白丝束。随后形成粘着斑在领先的优势，在后缘，驱动收缩和回缩肌动球蛋白结合的驱动单元格前进。6三维 (3D) 细胞迁移目前不很清楚，然而，最近的研究表明，三维迁移是明显不同于上述描述二维空间中的机制和可能由变质机制。例如，最近的研究由皮特et al。表明，ECM 的属性，3D 矩阵中的单元格可以伪足和 lobopodium 驱动的迁移机制之间切换。7细胞迁移是愈合反应的一个关键组件，因为 3D 模型的细胞迁移是伤口愈合过程中了解到各种刺激的生理反应的关键。虽然 2D 表面细胞洄游行为已被证明在 3D 矩阵迁移有较大差别，最新体外模型的细胞迁移在创面愈合过程中，包括既定的划痕检测，利用二维单层文化。5,6,7,8,9,10,11,12,13,14更最近开发的检测方法有利用 3D 的矩阵，但仍然培养细胞单层上矩阵，从而限制了能够真正复述三幅员的细胞环境中体内。15
本文所述方法的目的是研究细胞迁移在生理相关的 3D 模型，而不是二维的表面，以获得更健壮的理解迁移反应与应用刺激相关联。此方法允许为内激活或抑制途径与细胞迁移有关的因素 3D 纤维蛋白凝块基质细胞迁移的评价。纤维蛋白矩阵被选为这种方法由于纤维蛋白关键作用在早期阶段的伤口愈合;以下损伤，纤维蛋白凝块形成内伤口环境，遏制失血和充当支架为初始细胞浸润在组织修复和重建。2,16,17,18,19,20此外，纤维蛋白用于临床作为一个外科的密封胶，密封胶的组成已经离不开细胞迁移和最终愈合结果。先前的研究由 Cox et al。由不同纤维蛋白和凝血酶浓度为优化纤维蛋白组成的纤维蛋白凝块，研究碱性成纤维细胞迁移。在这些研究中，从塑料盘子上的纤维蛋白凝块的细胞迁移的量化。20在这里我们提出一种允许在 3D 纤维蛋白环境内的细胞迁移的定量方法。虽然在本议定书中具体描述的方法使用纤维蛋白，此模型可以容易地修改使用替代基质材料，作为所需，如胶原或其他 3D 矩阵。此外，我们目前使用的成纤维细胞球体在这种测定方法因为碱性成纤维细胞迁移到创面上是非常重要的细胞外基质合成和组织修复/改建。然而，在修复过程中中性粒细胞的过程是要迁移到创面上，其次是巨噬细胞的第一单元格类型。成纤维细胞然后开始渗入伤口环境后最初的损伤，在创伤愈合过程的增殖期初约 48 小时。19这种测定方法可以容易地修改，包括中性粒细胞、 巨噬细胞或其他细胞类型以评估纤维蛋白凝块结构的变化是如何影响这些类型的细胞迁移。21
若要实现这种测定方法，第一，成纤维细胞球体被形成使用先前描述的干细胞培养技术改造。22成纤维细胞球体随后转入 3D 纤维蛋白矩阵和产物然后可以轻松地监视和量化几天。本议定书考虑到 3D 的生理系统通过嵌入 3D 细胞球形内纤维蛋白矩阵，从而避免限制使用二维生长表面与细胞单层模型洄游行为所带来的相关性，而还允许高度控制在体外环境中的细胞行为的评定方法。

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Materials&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium, 4.5 g/L Glucose, w/ Sodium Pyruvate, w/out L-Glutamine</td><td>VWR</td><td>VWRL0148-0500</td><td>DMEM already containing L-glutamine can also be used</td></tr><tr><td>100mm Tissue Culture Dish, Non-Treated, Sterilized, Non-Pyrogenic</td><td>VWR</td><td>10861-594</td><td>Dishes of any size can be used for hanging drop culture&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum from USDA approved countries, heat inactivated, sterile-filtered, cell culture tested</td><td>Sigma-Aldrich</td><td>12306C-500ML</td><td></td></tr><tr><td>L-glutamine</td><td>Fisher Scientific</td><td>ICN1680149</td><td>Not needed if using DMEM that already contains L-glutamine&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin Solution stabilized, sterile-filtered, with 10,000 units penicillin and 10 mg streptomycin/mL</td><td>Sigma-Aldrich</td><td>P4333-100ML</td><td></td></tr><tr><td>Human Dermal Fibroblasts, neonatal</td><td>Thermo Fisher</td><td>C0045C</td><td>Can be replaced with other cell type of interest</td></tr><tr><td>21 G x 1 1/2&#039; needle</td><td>BD&amp;nbsp;</td><td>305167</td><td>22 G x 1 1/2 needles will also work</td></tr><tr><td>1 mL syringe</td><td>VWR</td><td>89174-491</td><td>Syringes of any volume can be used</td></tr><tr><td>Cell Culture Multiwell Plates, Polystyrene, Greiner Bio-One (Individially Wrapped) (48 wells)</td><td>VWR</td><td>82051-004&amp;nbsp;</td><td></td></tr><tr><td>Human Fibrinogen - Plasminogen, von Willebrand Factor and Fibronectin Depleted</td><td>Enzyme Research Laboratories</td><td>FIB 3</td><td>Can be replaced with matrix protein of interest</td></tr><tr><td>Human Alpha Thrombin</td><td>Enzyme Research Laboratories</td><td>HT 1002a</td><td>May not be necessary depending on matrix protein of interest</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Equipment&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSL2 cell culture hood</td><td></td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td></td><td></td><td></td></tr><tr><td>Inverted microscope with 10X objective</td><td></td><td></td><td></td></tr><tr><td>Centrifuge compatibile with 15 mL tubes</td><td></td><td></td><td></td></tr></tbody></table>

characterizing cell migration within three-dimensional in vitro wound environments

目前，大多数体外模型的伤口愈合，既定的划痕检测等，涉及研究细胞迁移及创面闭合二维的曲面上。然而，哪些体内伤口愈合发生的生理环境是三维的而不是二维。它变得越来越清楚的细胞行为大大在不同二维与三维的环境;因此，就需要更多生理有关体外模型研究细胞迁移行为在伤口闭合。此处所述的方法可以更好地反映生理条件比先前建立的二维划痕检测三维模型中的细胞迁移的研究。这个模型的目的是评价细胞产物经细胞迁移远离嵌入或反 migratory 因素纤维蛋白基质的球体身体检查。使用此方法，从一个三维矩阵椭球体内细胞产物可以观察和随时间通过明视场显微镜和分析球体的身体部位是很容易量化。Pro 洄游和/或抑制因素对细胞迁移的影响也可以在此系统中进行评估。这种方法给研究者提供一种分析在三维伤口关联矩阵体外细胞迁移的简单方法，从而提高关联性体外细胞研究之前，使用体内动物模型。

可以利用球体文化成功计算临和反洄游的代理对体外成纤维细胞迁移的影响 可以通过 72 h (图 1) 一段挂滴文化形成 3D 成纤维细胞球体。经过文化时期，球体嵌入凝块矩阵和成像使用明视场显微镜 (图 2)。最初的区域的球体 （图中描述了作为地区北临白色边框） 决定使用 ImageJ，用作参考点以确定随后的细胞产物?...

Watch this Scientific Journal Video about Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments at JoVE.com

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

本议定书的目标是评估 pro 和反 migratory 因素影响三维纤维蛋白矩阵内的细胞迁移。

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

characterizing-cell-migration-within-three-dimensional-vitro-wound

Research

JoVE Journal

Bioengineering

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

7.6K 次观看.  North Carolina State University and The University of North Carolina - Chapel Hill. 本议定书的目标是评估 pro 和反 migratory 因素影响三维纤维蛋白矩阵内的细胞迁移。

视频: Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

细胞侵袭和迁移过程的评价: 视频 Microscope-based 刮伤法与博伊登室法的比较

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

科研

癌症研究

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. ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

一种在96孔板表演细胞迁移/伤口愈合设备

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

发育生物学

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

生物工程

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

免疫与感染

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

医学

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

生物学

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

量化颗粒水凝胶生物材料内部和颗粒状水凝胶生物材料中的三维细胞迁移

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...