Name: characterizing cell migration within three-dimensional in vitro wound environments
Uploaded: 2017-08-16T13:00:00
Description: Watch this Scientific Journal Video about Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments at JoVE.com

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 2...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpolarized+signaling+reveals+two+distinct+modes+of+3D+cell+migration.">Nonpolarized signaling reveals two distinct modes of 3D cell migration.</a> J Cell Biol. 197 (3), 439-455 (2012).</li><li>Carlson, M. W., Dong, S., Garlick, J. A., Egles, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Engineered+Models+for+the+Study+of+Cutaneous+Wound-Healing.">Tissue-Engineered Models for the Study of Cutaneous Wound-Healing.</a> Bioengineering Research of Chronic Wounds. , 263-280 (2009).</li><li>Doyle, A. D., Wang, F. W., Matsumoto, K., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+topography+underlies+three-dimensional+fibrillar+cell+migration.">One-dimensional topography underlies three-dimensional fibrillar cell migration.</a> J Cell Bio. 184 (4), 481-490 (2009).</li><li>Fraley, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinctive+role+for+focal+adhesion+proteins+in+three-dimensional+cell+motility.">A distinctive role for focal adhesion proteins in three-dimensional cell motility.</a> Nat Cell Biol. 12 (6), 598-604 (2010).</li><li>Petrie, R. J., Doyle, A. D., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+versus+directionally+persistent+cell+migration.">Random versus directionally persistent cell migration.</a> Nat Rev. Mol Cell Biol. 10 (8), 538-549 (2009).</li><li>Rommerswinkel, N., Niggemann, B., Keil, S., Z&#228;nker, K. S., Dittmar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Cell+Migration+within+a+Three-dimensional+Collagen+Matrix.">Analysis of Cell Migration within a Three-dimensional Collagen Matrix.</a> J Vis Exp. (92), (2014).</li><li>Jonkman, J. E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+the+wound+healing+assay+using+live-cell+microscopy.">An introduction to the wound healing assay using live-cell microscopy.</a> Cell Adh Migr. 8 (5), 440-451 (2014).</li><li>L&#228;mmermann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Chen, Z., Yang, J., Wu, B., Tawil, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Three-Dimensional+Wound+Healing+Model.">A Novel Three-Dimensional Wound Healing Model.</a> J Dev Biol. 2 (4), 198-209 (2014).</li><li>Ross, R., Benditt, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+collagen+formation.">Wound healing and collagen formation.</a> The J Cell Biol. 12 (3), 533-551 (1962).</li><li>Sproul, E. P., Argraves, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytokine+axis+regulates+elastin+formation+and+degradation.">A cytokine axis regulates elastin formation and degradation.</a> Matrix Biol. 32 (2), 86-94 (2013).</li><li>Almine, J. F., Wise, S. G., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin+signaling+in+wound+repair.">Elastin signaling in wound repair.</a> Birth Defects REs C Embryo Today. 96 (3), 248-257 (2012).</li><li>Tracy, L. E., Minasian, R. A., Caterson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+and+Dermal+Fibroblast+Function+in+the+Healing+Wound.">Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound.</a> Adv Wound Care. 5 (3), 119-136 (2016).</li><li>Cox, S., Cole, M., Tawil, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+Human+Dermal+Fibroblasts+in+Three-Dimensional+Fibrin+Clots:+Dependence+on+Fibrinogen+and+Thrombin+Concentration.">Behavior of Human Dermal Fibroblasts in Three-Dimensional Fibrin Clots: Dependence on Fibrinogen and Thrombin Concentration.</a> Tissue Eng. 10 (5-6), 942-954 (2004).</li><li>Brown, A. C., Barker, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin-based+biomaterials:+Modulation+of+macroscopic+properties+through+rational+design+at+the+molecular+level.">Fibrin-based biomaterials: Modulation of macroscopic properties through rational design at the molecular level.</a> Acta Biomaterialia. 10 (4), 1502-1514 (2014).</li><li>Bartosh, T. J., Ylostalo, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+anti-inflammatory+mesenchymal+stem/precursor+cells+(MSCs)+through+sphere+formation+using+hanging+drop+culture+technique.">Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging drop culture technique.</a> Curr Protoc Stem Cell Biol. 28, (2014).</li><li>Bainbridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+the+role+of+fibroblasts.">Wound healing and the role of fibroblasts.</a> J Wound Care. 22 (8), 410-412 (2013).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="692">
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		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Materials</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Dulbecco&#39;s Modified Eagle&#39;s Medium, 4.5 g/L Glucose, w/ Sodium Pyruvate, w/out L-Glutamine</td>
			<td style="width:91px;">VWR</td>
			<td>VWRL0148-0500</td>
			<td style="width:217px;">DMEM already containing L-glutamine can also be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">100mm Tissue Culture Dish, Non-Treated, Sterilized, Non-Pyrogenic</td>
			<td style="width:91px;">VWR</td>
			<td>10861-594</td>
			<td style="width:217px;">Dishes of any size can be used for hanging drop culture&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Fetal Bovine Serum from USDA approved countries, heat inactivated, sterile-filtered, cell culture tested</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>12306C-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">L-glutamine</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td>ICN1680149</td>
			<td style="width:217px;">Not needed if using DMEM that already contains L-glutamine&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Penicillin-Streptomycin Solution stabilized, sterile-filtered, with 10,000 units penicillin and 10 mg streptomycin/mL</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Human Dermal Fibroblasts, neonatal</td>
			<td style="width:91px;">Thermo Fisher</td>
			<td>C0045C</td>
			<td style="width:217px;">Can be replaced with other cell type of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">21 G x 1 1/2&#39; needle</td>
			<td style="width:91px;">BD&#160;</td>
			<td>305167</td>
			<td>22 G x 1 1/2 needles will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">1 mL syringe</td>
			<td style="width:91px;">VWR</td>
			<td>89174-491</td>
			<td>Syringes of any volume can be used</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Cell Culture Multiwell Plates, Polystyrene, Greiner Bio-One (Individially Wrapped) (48 wells)</td>
			<td style="width:91px;">VWR</td>
			<td>82051-004&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Fibrinogen - Plasminogen, von Willebrand Factor and Fibronectin Depleted</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>FIB 3</td>
			<td style="width:217px;">Can be replaced with matrix protein of interest</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Alpha Thrombin</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>HT 1002a</td>
			<td style="width:217px;">May not be necessary depending on matrix protein of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Equipment</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSL2 cell culture hood</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell culture incubator</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Inverted microscope with 10X objective</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Centrifuge compatibile with 15 mL tubes</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></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?...

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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 ...

Research

JoVE Journal

Bioengineering

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Three-dimensional Optical-resolution Photoacoustic Microscopy

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...