这项工作是由来自美国国立卫生研究院（HL118532，HL120142，CA187692），大卫和露西尔·帕卡德基金会，卡米尔和亨利德雷福斯基金会和巴勒斯补助部分支持欢迎基金。 ASP是由夏洛特伊丽莎白宝洁尊敬的奖学金的部分资助。

1.溶液的制备<ol><li>以制备胰岛素的5毫克/毫升的溶液， 用蒸馏水用5mM盐酸（HCl）稀释粉末胰岛素库存（500毫克胰岛素在100ml溶剂）。制备100ml加入50μl浓HCl至100ml蒸馏水溶剂（DH 2 O）。 </li><li>使PBS的1倍溶液，稀释10倍磷酸盐缓冲盐水（PBS）原液在无菌条件下与卫生署2 O至1倍。 </li><li>制备聚二甲基硅氧烷（PDMS）弹性体溶​​液如下:1（重量:重量）的比例与PDMS的预聚物在10固化剂混合在一起。 </li><li>制备细胞培养基如下:向500 ml储备Dulbecco氏改良Eagle培养基中:营​​养混合物F-12（DMEM / F-12），加入10毫升无菌胎牛血清（FBS），500微升庆大霉素试剂，和500微升5毫克/毫升的胰岛素。 </li><li>以在1×PBS溶液制备1％牛血清白蛋白（BSA），加入1％（重量:V）BSA粉液为1x PBS调匀。 </li><li>以制备中和的牛I型胶原蛋白的一个4毫克/毫升的溶液，加入50微升10×Hank氏平衡盐溶液（HBSS）缓冲液，30微升的0.1N氢氧化钠（NaOH），30微升的细胞培养基，和400微升库存胶原在激冷1.5 ml离心管。吹打向上和向下慢慢搅拌;避免引入气泡。要删除任何气泡，短暂离心在4℃混合物。 </li><li> 4（V:V）在1X PBS稀释16％多聚甲醛1准备固定液。 </li><li>通过稀释股票核标签解决方案1准备核标签解决方案:1000（体积比）在1X PBS。 </li><li>洗涤剂:由1×PBS中的0.3％（体积v）的混合制备的0.3％的磷酸盐缓冲的盐水和洗涤剂（PBST）溶液。 </li><li>准备通过稀释山羊血清1点10分（体积:体积）封闭缓冲液在0.3％的PBST。 </li><li>通过稀释PRI制备用于感兴趣的特定蛋白质的初级抗体溶液玛丽抗体（例如，兔抗粘着斑激酶（FAK））1:200（体积比）在封闭缓冲液。 </li><li> （:v v）的封闭缓冲液1000:通过稀释的荧光探针缀合的抗体（例如，山羊抗兔）1制备二级抗体溶液。 </li></ol> 2.制备弹性邮票的三维微图案注意:弹性邮票与PDMS制成。 <ol><li>使在相同的比率的PDMS溶液如步骤1.3中所述调匀。放置在真空腔室中的混合物15-30分钟，以除去在混合过程中引入的任何气泡。 </li><li>倾脱气溶液到塑料称重舟或包含被光刻具有期望的几何形状的特征图案的硅主培养皿。为了达到最佳效果，治愈60℃的PDMS溶液〜12小时。 注:硅主人是高度可定制的;该协议采用矩形结构与尺寸50微米×200微米×50微米间隔200微米的距离。硅主人可以使用标准的光刻技术制成。简言之，将环氧类光致抗蚀剂旋涂在硅晶片的顶部。详述所需印章特征的掩模放置在光致抗蚀剂涂覆的晶片，然后将其暴露于UV光的顶部。没有被掩模所需要的特性被暴露于光，并在这些位置上的光致抗蚀剂变成交联的，而该光致抗蚀剂涂层的其余部分是可溶的，并且可以被冲走。 </li><li>后与PDMS已经围绕期望特征固化的硅母，从塑料容器中取出的PDMS和掌握。仔细分离从硅晶片与PDMS和从周围使用干净的刀片压印特征除去过量的PDMS。 </li><li>现在切断与压印特征成单个矩形邮票（〜8毫米×5毫米）用刀片图案化的PDMS。存储在这些CLE功能朝上一毫米直径100培养皿。 </li><li>此外，可使用在步骤1.3中所述的硅橡胶溶液，以使支撑件为矩形邮票。 </li><li>使用旋涂器，涂布在一个100毫米直径的培养皿〜2-3克的PDMS溶液均匀的，并在60℃下作为在步骤2.1固化〜12小时的PDMS。然后，用刀片，切的PDMS薄层成矩形（约5毫米×2mm）的。 注:需要在步骤2.3每笔PDMS邮票两个支撑。支撑可被存储在包含PDMS的邮票100毫米直径的培养皿。 </li><li>与PDMS邮票和载体可用于细胞培养之前，使用在生物安全柜吸气器（细胞培养罩）通过在70％乙醇和干浸没消​​毒。 </li></ol> 3. 3D上皮组织的制备<ol><li>在生物安全柜，大约50微升在PBS中的1％BSA的液滴添加到每张邮票的顶部。放置覆盖邮票液滴在4℃迷你4小时的妈妈，以确保的BSA吸附到印模表面。 </li><li>从PDMS邮票吸BSA解决方案。 </li><li>用细胞培养基（每洗涤50微升每邮票应足够）洗邮票的表面两次，每次洗涤后吸出。 </li><li>手柄采用弧形不锈钢镊子消毒PDMS邮票和支持。在生物安全柜，奠定了35毫米直径的一种组织培养皿每个PDMS邮票。在每个培养皿中，放下的距离比PDMS邮票的长度略小于分开的两个PDMS支撑。 </li><li>使用镊子，灭菌许多圆形盖玻片（15毫米直径，＃1），为有用70％的乙醇的PDMS邮票。吸从盖玻片多余的液体，同时与镊子保持它们。存放洗净盖玻片在一个单独的100毫米直径的培养皿。 </li><li>免除〜50微升胶原混合物的均匀涂层的每个的表面PDMS印模。 </li><li>捡起每个胶原涂覆PDMS压模与镊子和轻轻颠倒它们。降低对PDMS的顶部倒邮票支持布置在毫米直径35的组织培养抛出，使得胶原是印模和组织培养皿的底部之间。孵育的菜在37℃下30分钟。 </li><li>免除〜50微升剩余胶原混合物到每个圆形盖玻片（后，它们将被放置在工程化组织的顶部以完全封装它们在胶原蛋白），并在37°C孵育他们30分钟。 </li><li>在获取〜40％汇合含有上皮细胞（例如EpH4小鼠乳腺上皮细胞）一毫米直径100组织培养皿。吸出细胞培养基，并用10毫升1×PBS洗涤一次。 2毫升胰蛋白酶添加到细胞中，并在37°C孵育5-10分钟。 </li><li> 8毫升新鲜培养基的加入含有胰蛋白酶的细胞，从盘分离任何剩余的贴壁细胞的组织培养皿。通过移液轻轻混合和细胞混合物移至15毫升锥形管中并离心，在100×g下5分钟。 </li><li>吸从锥形管的上清，重悬在细胞培养基中细胞沉淀。使用血细胞计数器，计数细胞来确定悬浮液的浓度。调节悬浮液体积，得到10 6 -10 7个细胞/ ml的〜的终浓度。 </li><li>从培养箱中取出凝胶状胶原样品。使用镊子，轻轻抬起PDMS邮票径直向上从成型胶原蛋白分离他们丢弃。 </li><li>免除〜30微升浓缩细胞悬浮液在含所需的几何形状的模制腔各胶原凝胶的表面上。观察是10X / 0.25 NA目标的明显微镜下的细胞，同时轻轻摇晃的菜一边到另一边，以促进细胞的腔体内沉淀。空腔应该〜5分钟内被填充。 </li><li>在Remove过量细胞从空腔周围，在其一侧倾斜每个组织培养皿和胶原凝胶的表面上轻轻地免除〜400μl的细胞培养基。吸出液体，并重复洗涤1-2次，检查在显微镜下胶原凝胶在每次洗涤之间。 </li><li>后过量细胞已经从围绕在胶原模具填充细胞的空腔清零，放在组织培养皿到细胞培养孵化器在37℃下15分钟。然后，使用镊子，轻轻颠倒胶原涂层类盖玻片并将其放置在充满细胞胶原的模具，使得从盖玻片胶原形成在填充细胞腔的盖的顶部。孵育样品在37℃下15分钟。 </li><li>一旦胶原帽已经粘附到填充细胞胶原模具，慢慢分配〜2-2.5毫升细胞培养基通过在凝胶顶部的玻璃盖玻片。培养在37℃下1-3天的样品。 注意:最初的接种后24小时，在上皮组织可以与生长因子如表皮生长因子（EGF）或肝细胞生长因子（HGF）以诱导分枝进行处理。 </li></ol> 4.免疫荧光和图像分析<ol><li>吸从组织培养皿的细胞培养基，并添加足够的固定液以覆盖含细胞的凝胶。在室温下孵育的菜15分钟上以200rpm的摇床上。 </li><li>吸出固定液，填充用1×PBS的组织培养皿，并以200rpm在室温下孵育15分钟振荡器上。三总洗两次重复。 </li><li>从组织培养皿中吸出PBS并用Hoechst液代替:标记细胞核 。在室温下孵育15-20分钟。染色的斑或其他标记，它与抗体检测，跳到步骤4.5。 </li><li>吸出核标记溶​​液和洗涤含细胞接枝埃尔斯用1×PBS三次如步骤4.2。染色样品可以储存在1×PBS中在4℃直至进一步使用。 </li><li> 染色为目的蛋白质:从组织培养皿吸出PBS，加入300微升0.3％的PBST，并孵育在室温下将样品15分钟。 </li><li>吸出PBS，盖用封闭缓冲液的凝胶，并孵育在室温下〜4小时的摇床（200转）。 </li><li>吸出封闭缓冲液，盖上初级抗体溶液中的凝胶，并在4℃下在200rpm的摇床上孵育过夜。 </li><li>吸出第一抗体溶液，加入0.3％PBST溶液，并在室温下在200rpm的摇床上孵育30分钟。吸出PBST和重复每30分钟3-4小时。 </li><li>重复步骤4.7和4.8，此时用二级抗体溶液孵育。包裹组织培养皿用铝箔，以防止二次抗体的光漂白。国际泳联后l水洗，染色样品可以储存在1×PBS中在4℃直至进一步使用。 </li><li>为了形象化的样品，使用10X / 0.30 NA目标集中于上皮组织的平面。 </li><li>为了形象化细胞核，标记用在紫外光照射下一个10X / 0.30 NA目标的核标记图像固定样本。 </li><li>为了显现染色上使用倒置荧光显微镜10X / 0.30 NA物镜的所关注的蛋白质，图像样本。 </li><li>来创建相同的初始几何形状的多个组织（一般为50或以上）的蛋白质染色的频率图，第一​​进口分别使用标准图像分析软件的图像（见材料）并将其转换为8位灰度。 </li><li>阈值这些图像，通过定义一个灰度分界点把它们转换成二进制文件（ 即半色调/黑白）;低于阈值的灰度值变成黑色，并与上述的阈值变成白色。然后，结合每个单独的二进制图像成单个图像栈。 </li><li>接下来，注册堆叠图像（ 即对齐或与它们匹配）的分析软件。许多图像分析软件包都配有免费提供注册插件。 <ol><li>在一个堆使用每个图像作为下一图像的对准模板，以使得在整个堆叠中的图像由传播对齐。最后，覆盖（项目）的基础上平均强度在对准图像堆栈的图像，以形成与像素频率映射的单个图像。然后，该图像可以是彩色编码的使用选择10,11图像编辑软件。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

The protocol described above outlines a method to produce identical epithelial tissues of pre-defined shape, enabling spatial control of the mechanical stress experienced by cells in the tissue. An elastomeric mold is used to create cavities in type I collagen that are then filled with epithelial cells and covered with an additional collagen layer such that cells are completely encapsulated in a 3D collagen matrix environment. Further culture of these tissues and treatment with growth factors to induce branching from the...

支上皮组织，称为分支形态的发展，是由细胞衍生的，物理的和环境因素调节。在乳腺，分支形态是通过该引导集体细胞迁移创建树形结构一个迭代过程。第一步是从乳管主芽的形成，其次是分支起始和延长1,2。分支的侵入周围基质是由类固醇激素在青春期的全身性释放诱导。新的主芽，然后从现有分行的两端发起，而这个过程继续创造上皮树3。虽然许多重要的生化信号已经确定，指导这个复杂的发展过程，目前缺乏细胞生物学机制的全面了解。此外，在特定的线索的影响机理研究难以从experi解构体内 ments，因为精确时空扰动和测量常常是不可能的。 三维（3D）培养技术，例如全器官培养，主要类器官和细胞培养模型，是系统地研究组织形态4-6的基本机制的有用工具。这些可以用于确定特定因素的影响单独，例如机械力和生化信号，对各种细胞的行为，包括迁移，增殖和分化的是特别有用的。6工程化细胞培养模型，特别是容易使扰动的单个细胞和它们的微环境。 一个这样的养殖模式使用基于微细加工的方法来工程师控制的三维结构模型的乳腺上皮组织在与一个感应一贯和可重复形成集体迁移分支机构ppropriate生长因子。模型的主要优点是能够精确地操纵和测量的物理和生化因素，如机械应力的模式，具有很高的统计置信的影响的能力。该技术中，用计算模型一起，已经被用来确定物理和生化信号的乳腺上皮组织和其它支上皮7-11的正常发展的指导的相对贡献。这里介绍的是用于构建这些模型的组织，它可以被容易地扩展到其它类型的细胞和细胞外基质（ECM）凝胶的详细协议，其用作治疗剂的测试的潜在工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Ellsworth Adhesives</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS curing agent</td>
			<td>Ellsworth Adhesives</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithographically patterned silicon master</td>
			<td>self-made</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic weigh boat</td>
			<td>Fisher Scientific</td>
			<td>08-732-115</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm-diameter Petri dishes</td>
			<td>BioExpress</td>
			<td>D-2550-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol 200 Proof</td>
			<td>Pharmco-Aaper</td>
			<td>111000200</td>
			<td>Make a 70% EtOH (v:v) solution by mixing with dH2O</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>American Safety Razor</td>
			<td>620179</td>
			<td></td>
		</tr>
		<tr>
			<td>1:1 Dulbecco&#8217;s Modified Eagle&#8217;s Medium : Ham&#8217;s F12 Nutrient Mixture (DMEM/F12) (1:1)</td>
			<td>Hyclone</td>
			<td>SH30023FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Hank&#8217;s balanced salt solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>I6634-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Phosphate-buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP399-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>221465-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine type I collagen (non-pepsinized)</td>
			<td>Koken</td>
			<td>IAC-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from bovine serum (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved stainless steel tweezers</td>
			<td>Dumont</td>
			<td>7</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm-diameter tissue culture dishes</td>
			<td>BioExpress</td>
			<td>T-2881-6</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>BioExpress</td>
			<td>C-3394-2</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf Safe-Lock Tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular #1 glass coverslips, 15-mm in diameter</td>
			<td>Bellco Glass Inc.</td>
			<td>Special order</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% 1X Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>100503-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Perkin Elmer</td>
			<td>N9300260</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>HGF</td>
			<td>Sigma Aldrich</td>
			<td>H 9661</td>
			<td>Resuspended in dH2O at 50 mg/mL</td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse FAK antibody</td>
			<td>Life Technologies</td>
			<td>AMO0672</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa 488 antibody</td>
			<td>Life Technologies</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td>Used for color-coding pixel frequency maps.</td>
		</tr>
		<tr>
			<td>FIJI (ImageJ)</td>
			<td>NIH</td>
			<td>N/A</td>
			<td>Free image analysis software used for thresholding, registering, and overlaying images to create a pixel frequency map. The StackReg plugin was used for registering binary images.</td>
		</tr>
	</tbody>
</table>

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.

乳腺上皮组织微细化一般原理 的微细加工程序概述了实验工作流的一般原理图如图1所示。最终的结果是被完全嵌入的ECM凝胶中相同的几何形状和间距的上皮组织的阵列。代表性实验采用牛I型以4毫克/毫升的浓度胶原凝胶培养EpH4小鼠乳腺上皮细胞。为了确保工程组织的最高质量，在协议中列出的技术应密切随访。已被塑造成?...

Watch this Scientific Journal Video about Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix at JoVE.com

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

该原稿描述了软基于光刻技术工程师三维（3D）的均匀阵列由细胞外基质包围限定的几何形状的上皮组织。这种方法是适合于多种细胞类型和实验的上下文，并允许对于相同重复的高通量筛选。

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, ...

engineering-three-dimensional-epithelial-tissues-embedded-within

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.5K 次观看.  Princeton University. 该原稿描述了软基于光刻技术工程师三维（3D）的均匀阵列由细胞外基质包围限定的几何形状的上皮组织。这种方法是适合于多种细胞类型和实验的上下文，并允许对于相同重复的高通量筛选。 

视频: Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

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

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

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, 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 ...

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 ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

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 ...

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 ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

准备可调细胞外基质微环境，以评估施万细胞表型规范

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

在微加工微流体平台中生成简化的三维芯片皮肤模型

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...