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） 球体成熟时间可能必须加以调整，以获得一致的、 稳定的球体形成。慢慢地比成纤维细胞增殖的细胞类型，可能需要确保适当的球体形成 72 小时的时间内初始细胞浓度较高。细胞不应停留在挂滴文化长于 72 h 由于无力改变媒体对文化的球体。在初始的球体大小差异预计由于固有的生物学变异;我们解决这种可变性，球体形成入手细胞尽量减少大小差异尽可能多的一致数目以及恢复时间以供数据比较的增长。
此方法的实现面临的首要挑战是球体转移挂掉进血凝块矩阵的文化。本议定书使用针头和注射器抽吸球体包含液滴从倒置的培养皿盖子，但其他方法，如使用镊子或微附加到裁剪技巧，也可用于有效地转移从挂落到矩阵的球体。球体可以通过拉动注射器柱塞的可靠传输背稍前转移的液滴，并确保，液滴拟定入针慢慢让气泡不引入到微滴。注射器柱塞应只拉回足以把整个液滴带进针;拉回任何进一步将还介绍泡沫和干扰球成井的井板的可靠传输。专门的挂滴的文化板也可用于简化球体文化，却较少成本高效的手段这样做比简单地使用一个标准的菜。
在伤口愈合的体内，侵入伤口面积的成纤维细胞产生导致纤维蛋白基质降解的广泛的丝氨酸蛋白酶。这些细胞随后合成新的细胞外基质，主要由组成的胶原，以修复受损的组织。23这种迁移的机制允许控制入侵而不由我们的方法捕获和表示系统的局限性。此外应指出的是利用这种方法在成纤维细胞会分泌蛋白酶，能随着时间的推移会降解纤维蛋白网络;因此，如果长期 (&gt; 72 小时) 迁移研究期望，蛋白酶抑制剂，如抑肽酶，应添加到细胞培养基。
尽管有这些限制，此方法提供了简单、 性价比高，重现性好的方法，用来研究三维细胞迁移在体外。同时提供更多生理相关的环境比常用的二维模型，在此处所述的 3D 模型的增长分析维护 2D 模型所提供的直接分析。球体很容易就可以想象使用简单明视场或相显微镜和，如果需要，此方法可以方便地修改荧光显微镜用的荧光标记细胞生物膜和基质蛋白。此协议使用新生儿人真皮成纤维细胞成纤维细胞迁移在组织修复/重塑中的作用。如果需要，中性粒细胞、 巨噬细胞、 角质形成细胞或其他细胞类型可用于在这种测定方法评估纤维蛋白凝块结构的变化是如何影响其他伤口愈合过程中所涉及的单元格类型迁移。3D 材料可能也会根据需要进行修改;根据所需的应用程序，胶原或其他 3D 矩阵材料代替了纤维蛋白矩阵提供生理相关的模型。一个有用的模型为研究长期细胞迁移在伤口类似环境中的能获得与本议定书通过使用角质形成细胞和胶原合成，而不是成纤维细胞及纤维蛋白。所述的方法还允许更大的灵活性和成本效益，在细胞迁移和伤口愈合研究通过生理有关体外模型的实现，而不是要求立即体内工作继经典二维体外研究。

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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从球体机构的自由度。初始的平均面积测量的球体被确定为 4.3 x 104 μ m2 ± 1.1 x 104 μ m2为控制球体，4.4 x 104 μ m2 ± 1.1 x 104 μ m2的 pro 洄游扁球形和 4.5 x 104 ± 7.7 x 103 μ m2为反洄游的球体。包含或反 migratory 因素媒体被添加到球体包含凝块，然后从初始球体身体细胞产物最终图像原始媒体添加 (图 3) 后采取 72 小时拍摄过照片每隔 24 小时。
当实验完成后，分析了球体增长随着时间的推移，使用 ImageJ 来计算每个样本的球体区域的变化。如图 3所示，最外层的细胞群在每个时间点用于确定具体时间程度的细胞产物。我们期望，图 3所示的外的聚焦点是细胞碎片，我们不会算这在我们的量化。全尺寸图像显示清晰、 连续的单元格边框摆脱球体的身体;它是这追溯确定产物时的边界。细胞产物进行定量化正常化细胞生长面积相对于原始的球体身体区在 t = 0 h，然后用原始的球体区域的百分比增长。细胞生长在 72 小时期间定量揭示了球体接触到 pro 洄游的代理陈列迁移从原来的椭球与球接触到的身体程度有显著提高迁移抑制剂和控制球体暴露标准媒体 (p &#60; 0.0001，图 4)。相反，球体接触到反洄游的代理展出的迁移从原始的球体身体时控制球体相比，显著降低的程度 (p &#60; 0.0001)。
为了比较，我们提供示例划痕检测 (图 5)。这种测定方法进行播种成纤维细胞在 500,000 细胞/厘米2 24 井板 2 毫克/毫升血浆纤维蛋白原与涂层上。成纤维细胞被允许一夜之间长大。然后用移液管尖划破了井表面和细胞迁移到空闲区成像超过 24 小时。代表的形象，提出了一种在图 5A所示，对有限制这种测定方法;划痕不是重现性很好，在某些情况下，边界不清晰描绘。然而，整体的移民趋势在 pro 和反 migratory 因素是那些目前在 3D 球体的实验中，尽管有上述的变异性和迁移模式的差异一样。应该指出的是，迁移速度更快在 2D 的划痕实验中，相比 3D 球体测定。关闭最快在 pro 洄游剂存在下，培养在 pro 洄游剂存在下的所有样品被完全都关闭 24 小时内。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56099/56099fig1.jpg"> 
图 1: 人真皮成纤维细胞在挂掉文化。细胞被留在挂降为 72 小时，诱使球体形成的文化。顶部 (A) 和 (B) 侧视图的悬滴文化介绍。(C) 示意图描绘的悬滴球体形成的侧面图。黑色线代表的板顶部、 粉红色球表示媒体，和紫色领域表示的单元格内的球体。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56099/56099fig2.jpg"> 
图 2: 球体外观后初始嵌入在纤维蛋白矩阵 (t = 0 h 后添加介质) 成像使用明视场显微镜在放大 10 倍。（A、 D）控制球体;（B、 E）椭球暴露反移民代理在媒体;（C、 F）暴露在媒体 pro 洄游代理的球体。图像显示与 (D F) 和 (A-C) 没有白线，表明用于确定产物区域的边界。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56099/56099fig3.jpg"> 
图 3: 成像使用明视场显微镜在放大 10 倍的 72 小时后的球体外观。（A、 D）控制球体;（B、 E）椭球暴露反移民代理在媒体;（C、 F）暴露在媒体 pro 洄游代理的球体。图像显示与 (D F) 和 (A-C) 没有白线，表明用于确定产物区域的边界。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56099/56099fig4.jpg"> 
图 4: 球体增长超过 72 h.Pro 洄游和反移民代理在媒体列出可以明显影响碱性成纤维细胞迁移和增殖在 3D 环境中。N = 3/组;*表示 p &#60; 0.0001。误差线代表标准偏差。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig4large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56099/56099fig5.jpg"> 
图 5: 比较标准的 2D 划痕检测到。(A) 代表的形象，细胞迁移在标准的划痕检测超过 24 小时。成纤维细胞生长在 pro 或反 migratory 因素与条件培养基和划痕封闭的差异随着时间的推移进行量化。(B) %关闭时间过多，在控制，pro 洄游，或者反移民媒体上生长的成纤维细胞。虽然目前在此 2D 与 3D 球体法测定迁移模式的差异，整体迁移趋势保持一致。N = 3/组;*表示 p &#60; 0.05。误差线代表标准偏差。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig5large.jpg" target="_blank">请点击这里查看此图的大版本。</a>

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 ...

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Nanyang Technological University

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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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

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

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

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 ...

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

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 maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

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

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

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

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 ...

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

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

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

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

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

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

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

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

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

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

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

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

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 ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...