我们要感谢实验室的老成员, 帮助改善和完善这些技术的实际状态: Dr. 西莉亚。Dr. 安娜 Mrowiec, Dr., 拉肯纳和 Dr.-加西亚。我们感谢医院 Clínico 体育馆圣女 Arrixaca 大力支持这些技术的发展。还有干杯卡洛斯三世, 基金 de 门多萨市 Sanitarias。计划国家 i + D + i 和干杯研究所-Subdirección 总 de Evaluación y 调值研究 (批准号: PI13/00794);www.isciii.es. Fondos 菲德 "manera de hacer 欧罗巴"。我们还感谢穆尔西亚、IMIB-Arrixaca 和 FFIS 的行政支助和援助。最后, 我们要特别感谢 Dr. 伊莎贝尔-马丁内斯-Argudo 和 Facultad de Ciencias Ambientales Bioquímica, 校园 Tecnológico de Fábrica 武器, 卡斯蒂利亚 la 恰尔, 托莱多为他们的亲切的支持自愿割让生物医学和生物技术实验室, 使本论文的拍摄部分成为可能。

1. 定量研究的人工伤口划痕法
<ol>
	<li>细胞单层制备
	<ol>
		<li>在无菌条件下工作, 用血清补充培养基在培养瓶中种 Mv1Lu 或 HaCaT 上皮细胞。每24-48 小时刷新一次介质。在单元格达到80% 汇合后, 使用适当的方法分离单元格,即, trypsinization9。 注: Mv1Lu 和 HaCaT 上皮细胞系分别用 EMEM 和 DEMEM 培养基培养, 并辅以2毫米 l-谷氨酰胺, 在37° c 下孵育 5% CO2的受控气氛。每一个细胞线必须进行彻底的检测, 以确定细胞浓度和时间需要达到完全70-100。通常为 Mv1Lu 或 HaCaT 细胞, 2-4 x 104或 4-6 x 104单元格/好, 分别在 175 cm2培养烧瓶中播种, 在80% 汇合处的收益率高达 35 x 106 Mv1Lu 或 1 x 107 HaCaT 单元格。</li>
		<li>在12井或24井培养板, 种子在每井2毫升的细胞。每24-48 小时刷新一次介质, 确保电池数量足够高, 可以在2或3天后达到100% 汇合。</li>
		<li>细胞到达汇合处后, 取出血清补充培养基, 用新鲜无血清培养基冲洗两次。在刮伤前24小时将细胞保持在无血清培养基中。 注: Mv1Lu 或 HaCaT 细胞没有特殊的承印物要求;然而, 对于易在无血清条件下自发分离的细胞系, 使用聚 l-赖氨酸溶液的培养板表面的预应考虑为10。</li>
	</ol>
	</li>
	<li>单层刮伤
	<ol>
		<li>在无菌环境中工作, 通过牢固地拖动20µL 或200µL 无菌微尖端的窄端, 根据所需的划痕宽度, 从头开始培养单层。保持尖端与培养面垂直, 最大限度地提高间隙均匀性。<ol>
			<li>将培养皿放置在深色表面上, 以清楚地监视细胞单分子。如果需要, 在每个井上执行两个垂直划痕 (十), 以研究多达4迁移区。</li>
		</ol>
		</li>
		<li>通过轻轻去除培养基, 轻轻地加入新鲜无血清培养基, 洗涤分离细胞。重复两次, 或直到大多数未连接的单元格被移除。</li>
	</ol>
	</li>
	<li>实验程序和数据收集
	<ol>
		<li>采用一台倒置的相衬显微镜, 结合 CCD 相机, 以每井的划痕间隙为中心的4前处理参考图像。通过将显微镜场的边缘与划痕的相邻交叉点对齐, 选择感兴趣区域。 注意: 通常, 图像是使用10x 放大倍数和 1280 x 1024 像素图像大小获取的。如上所述, 选择感兴趣的区域有助于方便地定位和验证每井4测量。</li>
		<li>一旦所有的图像获得, 指定治疗井;用含有选定处理的新鲜培养基在井中交换培养基。 注: 另外, 对于不干扰培养基同质性的化学品或化合物, 可以直接将治疗接种到培养基中。</li>
		<li>孵育刮板 (37 ° c 与控制气氛包含 5% CO2), 直到观察到的条件达到90% 划痕间隙闭合。避免完全关闭。 注: 总间隙闭合使后处理图片收集无法检测, 无法确定间隙闭合的时间基准。在 Mv1Lu 或 HaCaT 细胞的情况下, 需要16-19 小时达到90% 汇合。</li>
		<li>通过固定细胞来阻止细胞迁移;在 PBS 中用1毫升4% 福尔马林轻轻地取代实验培养基。在 RT 处孵育15分钟。</li>
		<li>清洗细胞去除过量福尔马林;用新的 PBS 将溶液轻轻地替换在井中。重复至少两次。 注: 在这个阶段, 在密封之后, 板材可以被存放在4° c 无限期地。</li>
		<li>从划痕后捕获的原始参考区域中对每一个井后的参考图像进行处理。使用相同的设备 (倒置光学相衬显微镜, 结合 CCD 相机) 和匹配的图片设置 (放大和数字分辨率/密度)。 注意: 对于单独迁移并填补间隙的单元格, 独立于相干锋的形成 (例如, MDA-MB-231 乳腺癌人细胞), 时效图像可能允许计算单个细胞迁移速度。</li>
	</ol>
	</li>
	<li>图像分析与迁移量化
	<ol>
		<li>使用图像处理软件 (例如, ImageJ), 定义划痕间隙限制, 并确定在预处理图片中多达四测量的间隙表面。将数据记录为 "预处理间隙曲面" (PREGAP)。对后处理图片重复相同的过程。将数据记录为 "后处理间隙曲面" (POSTGAP)。<ol>
			<li>使用 ImageJ 打开录制的图像。在 "图像" 菜单中, 将图像类型设置为8位。在 "进程" 菜单中, 转到 "过滤器" 子菜单并应用 "方差" 过滤。</li>
			<li>在 "图像" 菜单中, 输入 "调整" 子菜单, 并将阈值设置为黑白 (B 和 #38; W), 确保未选中 "暗背景"。在 "进程" 菜单中, 转到 "二进制" 子菜单, 然后选择 "填充孔"。</li>
			<li>在 "分析" 菜单中, 选择 "设置测量" 并激活 "区域"。然后绘制在迁移边缘轮廓之后的可测量区域, 从而划定间隙。</li>
			<li>在 "分析" 菜单中, 选择 "分析粒子" 并记录绘制区域表面的 "总面积" 值。</li>
		</ol>
		</li>
		<li>在电子表格中引入 PREGAP 和 POSTGAP 的总面积值, 以量化每个样本集的绝对迁移, 作为间隙表面测量的差异: PREGAP − POSTGAP [任意单位]。另外, 将每个条件的绝对迁移数据规范化为控制样本: (采样/控制) * 100 [%]。 注意: 对于单独迁移和填充间隙的单元格, 独立于相干锋的形成, (例如, MDA-MB-231 的乳腺癌人细胞), 绝对迁移可以通过计数细胞侵入中心地带, 无论是在控制或处理过的样品。</li>
		<li>绘制量化输出 (请参见图 1)。必要时进行统计分析。</li></ol></li></ol>当比较两个条件或方差检验的分析时, 考虑学生的 t 检验, 以得到更多的条件。
	
	

2. 地形研究的人工迁移前沿试验
<ol>
	<li>细胞单层制备
	<ol>
		<li>在无菌条件下工作, 将一层消毒的圆形片放在空的培养基中, 直到盘子表面完全覆盖。 注: 最多12和33片适合5厘米和10厘米直径的板材, 分别。</li>
		<li>在培养基上, 在2或3天后, 轻轻地将足够数量的细胞聚集在一起, 使100% 汇合。确保没有片重叠相邻的片和防止片的浮动, 应用温和的压力与无菌微提示。每24-48 小时刷新介质。 注意: 每一个细胞线必须进行彻底的检测, 以确定细胞浓度和时间需要达到完全70-100。通常为 Mv1Lu 或 HaCaT 单元格, 分别为 2-3 x 106或 2.5-4 x 106 cells/10 cm 直径板。对于较小的板材, 必须缩小单元格数。</li>
		<li>细胞到达汇合处后, 取出血清补充培养基, 用新鲜无血清培养基代替。在进行人工伤口前, 将细胞保持在无血清培养基中24小时。 注: Mv1Lu 或 HaCaT 细胞没有特殊的承印物要求;然而, 对于易在无血清条件下自发分离的细胞系, 使用聚 l-赖氨酸溶液预的培养表面应考虑为10。</li>
	</ol>
	</li>
	<li>单层伤
	<ol>
		<li>使用无菌镊子, 轻轻地移动一个片到一个干净的10厘米板含有新鲜的无血清培养基。用镊子在外围片, 避免损坏单层。避免过度的压力, 可能导致片破损。</li>
		<li>通过在片中心的横线上拖动一条消毒的刀片来制造人为的伤口。拖3-4 毫米来回, 为了完全删除中央单层带。确保没有细胞残骸附着在受伤的边缘。 注: 在直径为12毫米的圆片上, 切口是在片的中部进行的。确保在主前侧留有足够大 (至少2毫米宽) 的细胞, 以避免片在以下步骤中倾斜。</li>
		<li>将受伤的片与面对向上的细胞转移到一个干净的6井板上, 其中含有至少2毫升新鲜无血清培养基。可容纳4受伤的片/井。</li>
		<li>用新鲜无血清培养基轻轻冲洗两次, 去除分离细胞。</li>
	</ol>
	</li>
	<li>实验程序和取样
	<ol>
		<li>用含有选定处理的新鲜培养基轻轻地在井中交换培养基。 注: 另外, 对于不干扰培养基同质性的化学品或化合物, 可以直接将治疗接种到培养基中。谨慎行事, 避免任何潜在的细胞脱离。</li>
		<li>保持板在细胞孵化器 (37 ° c, 5% CO2) 为期望的实验时间。</li>
		<li>通过固定细胞来阻止细胞迁移;在 PBS 中用1毫升4% 福尔马林轻轻地取代实验培养基。在 RT 处孵育15分钟。 注: 对于时间课程的实验, 从培养基中取出样品, 并将其固定在一个单独的盘子中, 以避免甲醛烟雾影响其他的, 正在进行的实验条件。</li>
		<li>清洗细胞去除过量福尔马林;用新的 PBS 将溶液轻轻地替换在井中。重复至少两次。 注: 在这个阶段, 在密封之后, 板材可以被存放在4° c 无限期地。</li>
	</ol>
	</li>
	<li>免疫荧光 (IF) 和拓扑分析
	<ol>
		<li>如在其他地方所描述的那样对个别片进行染色和成像3,4,11。<ol>
			<li>Permeabilize 在 PBS 的0.3% 海卫 X-100 溶液中浸泡片10分钟。</li>
			<li>使用以下阻断液块30分钟: 0.3% 牛血清白蛋白;10% 胎牛血清;5% 脱脂牛奶;0.3% 海卫 X-100 解决方案在 PBS。 注: 不含牛奶的堵塞液可提前准备并贮存在-20 ° c。</li>
			<li>在潮湿的室内, 在室温下孵育1小时, 在无牛奶的阻断溶液中稀释初级抗体。将片倒置在15µL 抗体溶液中, 以确保适当的分布。 注意: 抗体工作稀释是可变的, 将取决于使用的抗体。例如, 兔 anti-c-六月抗体需要1/100 的稀释。</li>
			<li>洗涤三次, 蘸片在0.1% 海卫 X-100 解决方案在 PBS。</li>
			<li>在无奶阻塞缓冲液中稀释的二次抗体中孵育片30分钟。 注意: 抗体工作稀释是可变的, 将取决于使用的抗体。例如, 山羊抗兔 (488) 需要1/400 稀释以获得最佳的分辨率。其他标记试剂, 如笔和 Hoechst-33258 可以添加到孵化缓冲在这个阶段。</li>
			<li>洗涤三次, 蘸片在0.1% 海卫 X-100 解决方案在 PBS。</li>
			<li>将片倒置在10µL 安装介质放置 (例如, Vectashield) 放置在显微镜幻灯片上。在夜间将幻灯片留在室温下, 以使安装介质硬化。之后, 在黑暗中无限期地存放在4° c。</li>
		</ol>
		</li>
		<li>获得荧光或共聚焦显微镜图像的结构变化发生在迁移前边缘。 注意: 拓扑研究可以通过平铺图片从迁移前到内单层。通过基于软件的局部荧光强度分析, 可以进行半定量研究。</li>
	</ol>
	</li>
</ol>

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M., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Migration: Developmental Methods and Protocols. , 97-110 (2011).</li><li>Nyegaard, S., Christensen, B., Rasmussen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+accurate+quantification+of+cell+migration+using+human+small+intestine+cells.">An optimized method for accurate quantification of cell migration using human small intestine cells.</a> Metabolic Engineering Communications. 3, 76-83 (2016).</li><li>Szybalski, W., Iyer, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinking+of+DNA+by+Enzymatically+or+Chemically+Activated+Mitomycins+and+Porfiromycins,+Bifunctionally+"Alkylating"+Antibiotics.">Crosslinking of DNA by Enzymatically or Chemically Activated Mitomycins and Porfiromycins, Bifunctionally "Alkylating" Antibiotics.</a> Fed Proc. 23, 946-957 (1964).</li><li>Tomasz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitomycin+C:+small,+fast+and+deadly+(but+very+selective).">Mitomycin C: small, fast and deadly (but very selective).</a> Chem Biol. 2 (9), 575-579 (1995).</li><li>Sherry, D. M., Parks, E. E., Bullen, E. C., Updike, D. L., Howard, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+using+silicone+elastomer+masks+for+quantitative+analysis+of+cell+migration+without+cellular+damage+or+substrate+disruption.">A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption.</a> Cell Adh Migr. 7 (6), 469-475 (2013).</li><li>Dennis, E. A., Rhee, S. G., Billah, M. M., Hannun, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phospholipase+in+generating+lipid+second+messengers+in+signal+transduction.">Role of phospholipase in generating lipid second messengers in signal transduction.</a> FASEB J. 5 (7), 2068-2077 (1991).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li></ol>

作者声明没有利益冲突。

在皮肤或粘膜的破坏, 屏障功能恢复的行动, 许多细胞类型, 包括成纤维细胞或上皮和免疫细胞。共同, 这些细胞经历了一个复杂的过程, 涉及细胞凋亡, 增殖, 分化, 重要的是, 成纤维细胞和上皮组织迁移, 这是最终的机制, 负责修复被破坏的组织和浅上皮间隙闭合1,12因此, 研究细胞迁移有助于精确描述上皮细胞的生理行为, 同时也确定伤口治疗的调节电位?...

细胞迁移是需要的伤口愈合, 因为它是负责最后关闭的上皮间隙和恢复破坏表面1。在动物模型中执行人工伤口允许在近生理条件下复制这个复杂的过程2。然而, 这种方法往往导致昂贵和复杂的实验程序, 可能缺乏精确的研究不同的过程, 由于复杂的性质, 伤口愈合过程。
体外上皮细胞系的培养为研究这些细胞在创伤愈合中所起的作用以及治疗对细胞迁移行为的影响提供了一种有益的替代动物模型。利用 non-confluent 培养的分子技术, 经常研究上皮细胞的生理特性3,4,5,6;然而, 上皮完整性的破坏通常是通过细微的机械切口来实现的。在细胞培养中, 这意味着可忽略的细胞数可能暴露在伤口间隙, 它们代表了一个太小的样本分子生物学技术。然而, 这些病变可以在显微镜下进行研究, 利用一些上皮细胞系的固有迁移特性, 如水貂肺上皮细胞 (Mv1Lu) 或自发永生化的人角质形成细胞 (HaCaT)。线.
在这里, 我们描述了一种显微镜方法, 它是适合获得定量数据的上皮细胞迁移在伤口愈合的情况下3,4,7,8。此外, 我们提出的其他方法, 有助于研究的定性分子和形态学的变化发生在上皮膜的迁移。总的来说, 这些方法提供了一个框架来研究的动力学和形态学的变化涉及上皮细胞的行为和反应的治疗过程中伤口愈合。

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Biowest, Nuaill&#233;, France</td>
			<td>L0102-500</td>
			<td>Optional 10 % FBS supplement</td>
		</tr>
		<tr>
			<td>Eagles&#8217;s Minimum Essential Medium (EMEM)</td>
			<td>Lonza</td>
			<td>BE12 -662F</td>
			<td>Optional 10 % FBS supplement</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td>Use at 2 mM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific, Waltham, MA USA</td>
			<td>DE17603A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>T4049</td>
			<td>Dilute as appropriate</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>P9155</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS) (10x)</td>
			<td>Gibco by Life Technologies</td>
			<td>14200-067</td>
			<td>Dilute to 1x</td>
		</tr>
		<tr>
			<td>24-well culture plates</td>
			<td>BD FALCON//SARSTED</td>
			<td>734-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>SARSTEDT</td>
			<td>83-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>E9644</td>
			<td>Used 10 ng/mL</td>
		</tr>
		<tr>
			<td>Round cover glass</td>
			<td>MENZEL-GL&#196;SER</td>
			<td>MENZCB00120RA020</td>
			<td>SHORT DEPTH OF FIELD</td>
		</tr>
		<tr>
			<td>Reinforced razor blade no. 743</td>
			<td>Martor (through VWR)</td>
			<td>MARO743.50</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l sterile aerosol pipet tips</td>
			<td>VWR</td>
			<td>732-0541</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;l sterile aerosol pipet tips</td>
			<td>VWR</td>
			<td>732-0528</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera coupled phase contrast microscope</td>
			<td>Motic Spain</td>
			<td>Moticam camera 2300 3.0 M Pixel USB 2.0; Motic Optic AE31</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>ZEISS Microimaging, Germany</td>
			<td>LSM 510 META</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Culture dish</td>
			<td>BD FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti c-Jun antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1694</td>
			<td>Used 1:100</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG (polyclonal goat ) AF 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Used 1:400</td>
		</tr>
		<tr>
			<td>Hoechst-33258</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>14530</td>
			<td>Used 1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 phalloidin (in methanol) (red)</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>Used 1:100</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2323</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>BD DIFCO</td>
			<td>232100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health, USA</td>
			<td>Release 1.50i</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen LSM 510 image processing software</td>
			<td>ZEISS Microimaging, Germany</td>
			<td>Release 5.0 SP 1.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscopy based methods for the assessment of epithelial cell migration during in vitro wound healing

细胞迁移是伤口愈合的一个必需的方面。在研究动物模型上制造人工伤口往往导致昂贵和复杂的实验程序, 但可能缺乏精确性。体外上皮细胞系的培养为研究创面愈合过程中细胞迁移行为和治疗对这些细胞的影响提供了一个合适的平台。上皮细胞的生理学在 non-confluent 条件下经常被研究;然而, 这种方法可能不像自然伤口愈合的条件。通过机械手段破坏上皮细胞的完整性产生了一个现实的模型, 但可能会阻碍分子技术的应用。因此, 显微技术是最理想的研究上皮细胞迁移的体外.在这里, 我们详细介绍了两种具体的方法, 人工伤刮法和人工迁移前分析法, 可以分别获得定量和定性的数据, 对上皮细胞的迁移性能。

定量研究的人工伤口划痕法: 评估表皮生长因子 (EGF) 促进迁移:
EGF 是一个众所周知的诱导上皮细胞增殖和迁移, 从而对量化迁移促进的积极控制。Mv1Lu 和 HaCaT 细胞单分子膜用于创面划伤的检测, 并获得了预处理图像。在接种 10 ng/毫升 EGF 后, 细胞孵育19小时前固定和后处理图片。通过维持血清饥饿状况, 使其增殖保持在最低限度。?...

Watch this Scientific Journal Video about Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing at JoVE.com

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

本手稿描述了如何在培养的上皮细胞单分子膜切口样病变, 方便模型伤口愈合在体外, 允许成像的共焦或激光扫描显微镜, 并能提供高质量定量和定性数据, 用于研究细胞行为和参与迁移的机制。

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 ...

microscopy-based-methods-for-assessment-epithelial-cell-migration

Research

JoVE Journal

Medicine

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

9.2K 次观看.  IMIB-Arrixaca. 本手稿描述了如何在培养的上皮细胞单分子膜切口样病变, 方便模型伤口愈合在体外, 允许成像的共焦或激光扫描显微镜, 并能提供高质量定量和定性数据, 用于研究细胞行为和参与迁移的机制。

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

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

科研

医学

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

多式联运定量相成像数字全息显微准确评估肠道炎症和上皮伤口愈合

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

选择性化学腰椎交感神经切除术中灰色拉米关节的靶向性

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

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 ...