Name: engineering three-dimensional epithelial tissues embedded within extracellular matrix
Uploaded: 2016-07-10T14:00:00.000+00:00
Duration: 8 min 49 s
Description: Watch this Scientific Journal Video about Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix at JoVE.com

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 initial architecture make this system amenable for the study of branching morphogenesis of epithelial cells. There are several critical steps in the protocol. The first is lifting the PDMS molds straight upwards from the gelled collagen prior to cell seeding. Any horizontal movement during this step will distort the cavities, and they will no longer retain the desired geometry. Then, after the cavities have been seeded with cells, it is important to carefully wash off any excess cells that remain on the surface, as these may interfere with the behavior of the patterned tissues, particularly if they are very close to the tissues. Washing too rigorously, however, may result in cells being washed out of the cavities. Lastly, in the final micropatterning step when the cell-containing collagen gels are being immersed in culture medium, it is important to add the medium slowly and directly on top of the coverslip. Adding medium to the side of the dish away from the coverslip may result in detachment of the coverslip and the collagen lid. Each of these steps needs to be performed with care to achieve the best results.
Over the past few decades, several culture models have emerged for the study of epithelial morphogenesis, many of which involve the ex vivo culture of intact organs or explants of organs. Major advantages of culturing tissue taken directly from an animal are that tissue architecture remains intact and that cell-cell interactions are maintained. However, these models are not readily amenable to studies investigating the specific effects of a particular cell type or physical microenvironment. Additionally, organs and organ explants can be fragile and difficult to culture ex vivo. Others used primary mammary epithelial cells12, in which gene expression can be more readily manipulated, though this method does not conserve in vivo tissue architecture. Moreover, like intact organs, these cells can also be difficult to culture for long periods of time5. There is a need, therefore, for more robust culture models of epithelial morphogenesis that can be more precisely controlled and readily perturbed to gather reproducible data. 
A widely used 3D cell culture technique is the formation of epithelial cell clusters embedded in collagen or other ECM proteins11,13. Disadvantages of this technique, however, include the inability to precisely control or predict the location of clusters within the gel, cluster size and shape, and branching sites. In addition, it is difficult to manipulate or measure the mechanical cues experienced by cells in different regions of the tissue clusters. Engineered tissues enable better control of these parameters.
The engineered tissue protocol described here is an easy to use, versatile, and reproducible 3D cell culture method that eliminates the heterogeneity commonly found in ex vivo culture systems and 3D clusters. Such engineered cell culture models provide a platform for more precise control of physical and biochemical signals, and results can be easily combined with computational models to predict the effects of perturbations or measure cellular forces using techniques such as traction force microscopy (TFM)9,10. A caveat of the model system, however, is that it does not completely replicate the cellular, chemical, and physical microenvironment within the mammary gland in vivo. Nonetheless, essentially all parts of the system are modifiable: including the cells used (in which gene expression can also be altered by transfection), tissue geometry, ECM composition, and other biochemical factors (ex. growth factors). Primary mammary epithelial cells can also be cultured in this system; they form a lumen and undergo branching morphogenesis from the same locations as immortalized cell lines7. Moreover, the model can be modified to incorporate other cell types to more appropriately represent the native mammary gland microenvironment14, or to investigate the underlying mechanisms that give rise to any branched organ.

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><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 dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Razor blade</td><td>American Safety Razor</td><td>620179</td><td></td></tr><tr><td>1:1 Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium : Ham&amp;rsquo;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&amp;rsquo;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 dH&lt;sub&gt;2&lt;/sub&gt;O 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。已被塑造成一种矩形井阵列图2A和2B展示低和高放大倍率的意见胶原蛋白之前，细胞接种凝胶。孔的形状是由在硅主特征的形状决定。它解除个很重要ÈPDMS模具从胶原直线上升以免扭曲腔的几何形状。 图2C示出了在型矩形孔I型胶原已填充有乳腺上皮细胞（过量的细胞已被洗掉了胶原的表面）的凝胶。在本实施例中，每个为200μm×50微米的矩形孔含有大约80-100的细胞。 生长因子加成诱导的形态发生 接种24小时后，将组织可以与生长因子如HGF或EGF和几天培养模型分支形态进行处理。典型地，分支开始早生长因子刺激后4小时形成。 图3A示出了从一个类型内的矩形乳腺上皮组织的代表性结果I型胶原凝胶中的微细加工程序后24小时，在这之后CELLS已附着到胶原和彼此。无分支之前生长因子除了观察到。 图3B示出了经历了另外的HGF在10ng / ml的24小时后的分支的代表性矩形组织。在这种情况下，出现在组织的端部（相对于中间），其中，所述细胞经历的最高机械应力9分支。在相同的凝胶相同的初始几何形状的多个组织然后可以成像以确定分支位置和分支长度的人口平均，使高通量分析。 免疫荧光染色的可视化蛋白定位 在养殖模式的组织阵列的免疫荧光染色使我们能够确定具有较高的统计置信组织内蛋白质的定位。 图4a显示repre从染色粘着斑激酶（FAK）的支矩形乳腺上皮组织表性的结果。创建相同的几何形状的组织的频率映射可以用于可视化的组织中感兴趣的蛋白质，它可以比其它蛋白质的定位以及支活动的平均空间定位。 图4B示出平均FAK染色的50组织表示在长方形的组织，其中，支化，通常会发生的短端FAK富集的频率图。 <img alt="图1" src="/files/ftp_upload/54283/54283fig1.jpg" /> 图1. 原理概述了微加工过程。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54283/54283fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2.在微细加工拍摄的图像。（A）低和（B）与2型矩形腔的高倍率相差图像I型胶原使用弹性模PDMS创建。 （C）从（A）和（B）的空腔填充有乳腺上皮细胞。比例尺，100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54283/54283fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54283/54283fig3.jpg" /> 图3. 微构组织离岗分支形态:（A）的微细加工后的代表矩形组织24小时的相衬图像。 （B）一种重的相衬图像已经开始经受增加的HGF在10ng / ml的经过分支24小时表象矩形组织。白色箭头指示新组建的分支机构。比例尺，100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54283/54283fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54283/54283fig4.jpg" /> 图4. 免疫微构组织的染色。分支开始后乳腺上皮组织（A）免疫荧光染色FAK。 （ 二 ）组织50平均FAK染色的频率地图。比例尺，100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54283/54283fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

科研

癌症研究

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

纤维细胞衍生的三维矩阵系统适用于内皮管形成测定

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

生物工程

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

在开顶器官芯片上重建人上皮组织的细胞结构和功能

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

<meta charset="utf8"></head><body>用于上皮细胞和成纤维细胞的先进双层 3D 共培养系统

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

<meta charset="utf8"></head><body>作者聚焦:使用 PVA-PCL 纳米纤维膜推进 3D 共培养系统

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

使用纳米纤维膜建立上皮细胞的三维共培养模块

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...