感谢慷慨提供MIN6细胞克丽斯蒂Anseth的科罗拉多大学的实验室。这个项目的资金已经由美国国家科学基金会提供。

1。 PEGDM合成的光敏PEGDM单体溶液的配制<ol><li>一个用硬塑料盖40ML玻璃小瓶，称取PEG的2G（线性，万大）。 </li><li>加入约308μL甲基丙烯酸酐和松散的第小瓶。 </li><li>微波在一个标准的家用微波炉2分钟高小瓶。服装耐热手套，彻底涡旋小瓶，然后在​​高微波5分钟。 </li><li>简要允许小瓶不够冷静，要带手套处理。摘帽，并慢慢加入二氯甲烷，而纷飞的小瓶。加入足够的解决方案，使出现明确和同质化，需要震荡样品。亚甲基氯化物的总体积应为1.5 - 2毫升。 </li><li>在200毫升冷沉淀PEGDM，搅拌加入溶液滴加乙醚。 </li><li> PEGDM真空过滤收集，并允许它干。 </li><li>溶解于适当数量的PEGDM去离子水（通常为100 -150毫升）。 </li><li> 5天透析管透析千大分子量切断对去离子水的解决方案。每天更换一次水。 </li><li>分装到适当的容器内，并冻结在-80 ° C过夜的解决方案。冻干4天的解决方案，以产生一个纯净的白色PEGDM粉末。保存在4 ° C时不使用粉末。 </li><li>要准备一个光敏PEGDM解决方案，首先准备一个0.5重量％的光引发剂原液溶解在去离子水Irgacure 2959。涡的解决方案，并存储在黑暗中。 </li><li>准备了10％PEGDM和0.025％Irgacure 2959汉克的平衡盐溶液（HBSS）的解决方案。涡彻底，以确保PEGDM解散。我们通常准备时间的这一解决方案1ML。 </li><li>消毒注射器过滤器通过0.2微米到1.8 ml离心管过滤解决方案。包装在铝箔管并储存于4 ° C </li></ol> 2。文化，感染和MIN6细胞的聚合<ol><li> MIN6细胞的RPMI 1640与10％FBS，7mm的葡萄糖，100个单位/ mL青霉素/链霉素，0.5微克/毫升amphotercin乙在湿润的环境在37 ° C和5％的CO 2的培养基中培养。 </li><li>要释放从处理过的培养瓶表面的细胞，吸出培养基，冲洗细胞与37 ° C钙/镁免费的HBSS，吸的HBSS，再加入2毫升37 ° C胰蛋白酶- EDTA溶液，并允许细胞孵育3分钟。 </li><li>牢固JAR烧瓶的侧敲的细胞，然后加入8毫升37 ° C介质，大力移液器细胞悬液上下注意不要引入气泡。 </li><li>移液器无菌的15 mL离心管中的细胞，并使用血球或其他细胞计数过程进行细胞计数。 </li><li>在准备与制造商的感染细胞病毒，首先要确定被感染的细胞总数，计算量要求达到的所需的感染复数（MOI）（50％至100％传染性颗粒细胞）的病毒悬液。 </li><li>稀释分装在1.8 ml离心管体积的HBSS中前仔细pipeting适量病毒悬液在HBSS中的病毒。 HBSS中聚合的细胞，每孔50μL是适当的。 </li><li>驱散pipeting他们在他们管，然后吸管必要的量，实现成一个6孔悬浮培养板的孔，每孔40细胞的细胞。 </li><li>每孔加入适当稀释的病毒量，以达到所需的教学语言。 </li><li>中等至每口井，使总体积为1.7毫升。 </li><li>将盘上轨道摇床培养箱内。振动筛上设置一个较低的设置，使中等轻轻洗井（〜100 RPM）左右。允许细胞以aggregaTE为24-36小时。应形成一个直径约75-175微米聚合。 </li></ol> 3。细胞封装在PEGDM <ol><li>细胞可作为分散（步骤2.4）或以下的聚合（步骤2.10）封装。由于水凝胶的易制毒化学PEGDM解决方案是一种液体，细胞可能倾向于解决之前，交联和凝胶的形成。如果解决有望出现迅速，如较大的聚集，它可能是有益的两个步骤中产生凝胶，使细胞定居到中央的凝胶平面。一个单步水凝胶的合成过程，适合于分散的细胞显示（ 图 2），以及两个步骤所示（ 图 3），下面详细（3.2-3.9 ）。 </li><li>创建一个将削减1毫升的塑料注射器的锥尖，并把打开它在机架年底形成的水凝胶的船只。 </li><li>吸取20μL的光敏PEGDM，以便到柱塞注射器开放lution确保该卷涵盖了整个柱塞表面。调整柱塞以小增量，实现了平坦的液体表面。 </li><li>放置在机架中的位置下方约8分钟的紫外灯（365nm的，7〜毫瓦/平方厘米）机架，让水凝胶交联的吸管。在这段时间内，制备的细胞可能会开始</li><li>吸取含有1.8 ml离心管细胞/聚合所需数量的卷，第，130克，5分钟离心管。 </li><li>使用移液器，小心地取出，而不会干扰细胞沉淀在管尖媒体。由于媒体负责人沉淀的方法，它可能是有益的，使用更精细的10微升吸管尖。 </li><li>轻轻重悬细胞沉淀在20μL光敏PEGDM解决方案（使用宽口吸管尖，如果与聚合工作），并添加顶部的Th细胞悬液的注射器Ë以前交联水凝胶的一部分。 </li><li>注射器暴露的紫外光一个额外的8分钟内完成建设的水凝胶。完成后，细胞被完全封装外圆内水凝胶（ 图3）。 </li><li>弹出从注射器到1毫升以及一个24孔板中，简单地洗凝胶的HBSS的水凝胶。凝胶在24孔板和文化以及传输所需的条件下，以1毫升的媒体。 </li></ol> 4。缺氧跟踪<ol><li>在任何一点，在文化，形象的细胞用荧光显微镜来跟踪开始缺氧的信号。成像是什么，你可以多孔板的图像或凝胶转移到载玻片事先安置在显微镜舞台上。山顶激发的Ds红在556nm达到峰值发射发生在586nm。排放量是相当激烈，因此漂白尚未观测教育署是有问题的。启动蛋白质生产和第一信号检测之间的滞后时间约为12小时。信号是不够具体，识别单个信号细胞，但分辨率可能不足以确定细胞内信号本地化。在信令细胞，通常观察到的信号是在整个细胞质中均匀。还可以增加信号强度，长期暴露在缺氧，虽然我们尚未完全确定，如果这是由于蛋白表达增加，总蛋白的积累，或延迟蛋白成熟。 </li></ol> 5。代表性的成果封装MIN6聚集在PEG水凝胶的一个有代表性的例子是如图4所示。交联凝胶将固体整个反应的船只的形状。一个光滑的外表面的凝胶是可取的，有助于防止异物重新植入响应。凝胶内，应完全封闭的细胞聚集在基质中均匀分布，以便更好的养分运输。 MIN6聚集在缺氧的信号代表图像是描绘在图5。无论是20％O 2（5A）或1％O 2的 44小时（5B）相同MIN6标记病毒感染聚集培养前的图像采集。 <img alt="图1" src="/files/ftp_upload/3521/3521fig1.jpg" /> 图1。缺氧标记系统的活动的示意图。的DS红DR基因和上游的HRE启动子的腺病毒插入允许对缺氧诱导的HIF - 1的控制之下的荧光蛋白的生产。 <img alt="图2" src="/files/ftp_upload/3521/3521fig2.jpg" /> 单步封装程序流程图图2。分散在CR MIN6细胞osslinked PEGDM水凝胶。对于每个凝胶，分散的细胞悬浮在光敏PEGDM单体溶液40μL。这是摆在断头1ml注射器和365nm的紫外线光10-12分钟后下形成凝胶。工程完成后，水凝胶磁盘是从注射器中删除，洗净，放置在装满一个中等孵化孔板。 <img alt="图3" src="/files/ftp_upload/3521/3521fig3.jpg" /> 图3双步汇总MIN6细胞在交联PEGDM的水凝胶的封装程序流程图。对于每个凝胶，半凝胶是第一所形成的光敏PEGDM 8分钟的单体溶液20μL的紫外光交联。仔细暂停MIN6聚集在光敏单体的解决方案，这是预先形成的半凝胶上添加额外的20μL。凝胶形成与聚合的紫外线照射一个额外的8分钟，完全封装在内侧平面凝胶。 <img alt="图4" src="/files/ftp_upload/3521/3521fig4.jpg" /> 图4。40μL水凝胶20X放大倍率下的图像。 MIN6聚合（〜40个细胞）内的凝胶清楚地看到。水凝胶的直径约6毫米（栏= 1mm）的。 <img alt="图5" src="/files/ftp_upload/3521/3521fig5.jpg" /> 图5荧光缺氧信号汇总MIN6细胞在PEGDM凝胶。封装和20％O 2，然后放入孵化44小时的细胞不显示，而细胞，然后封装在2％的O 2 44小时孵育显示效果清晰，无处不在的信号的缺氧信号（一）。 （标尺示100μm）（B）。

<ol>
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没有利益冲突的声明。

这里介绍的方法，提供了一个细胞封装在非生理条件下的最小使用PEG凝胶快速和简单的技术。 PEG代表一个非常有用的封装材料，其生物相容性和易于修改。简单的PEG在光敏溶液的百分比变化，例如，可用于调节通过孔径的机械性能，如压缩模量和输运性质。此外，PEG很容易修改，除了侧链。聚乙二醇水凝胶，因此，是一种很有前途的临床设备和灵活的平台， 在体外研究跟踪PEG封装细胞缺氧的方法也被提出。缺氧检测的简单性和避免需要的利益而牺牲的细胞，这种方法非常有用。该技术可应用于多种类型的细胞在其我们的各种条件efulness广泛。例如，作为干细胞分化的提示缺氧可能是干细胞微团培养跟踪。但是，这种方法只能适用于分散的电池系统或系统中，分散细胞后汇总。此外，检测荧光信号可能难以在更大或更密集的组织。

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选 </td></tr><tr><td>聚乙二醇</td><td> Sigma - Aldrich公司</td><td> 309028 - 500G </td><td></td></tr><tr><td>甲基苯酐</td><td> Sigma - Aldrich公司</td><td> 276685 - 100ML </td><td></td></tr><tr><td>微波</td><td>艾默生</td><td> MW8784SB </td><td></td></tr><tr><td> Vortexer </td><td>科学工业</td><td> SI - A236 </td><td></td></tr><tr><td>二氯甲烷</td><td> Sigma - Aldrich公司</td><td> D65100 - 1L </td><td></td></tr><tr><td>乙醚</td><td> Sigma - Aldrich公司</td><td> 346136 - 1L </td><td></td></tr><tr><td>透析管</td><td>频谱</td><td> 132640 </td><td></td></tr><tr><td></td><td>实验室</td><td></td><td></td></tr><tr><td>冰箱</td><td></td><td></td><td></td></tr><tr><td&gt;冻干机</td><td> Labconco </td><td> 7670521 </td><td></td></tr><tr><td>真空泵</td><td>韦尔奇</td><td> 8917Z - 01 </td><td></td></tr><tr><td> Irgacure 2959 </td><td>汽巴嘉基</td><td> 029891301PS04 </td><td></td></tr><tr><td> HBSS </td><td> Mediatech </td><td> 21 - 022 - CV </td><td></td></tr><tr><td>注射器过滤器</td><td> VWR </td><td> 28145-477 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Mediatech </td><td> * </td><td> *自定义的制定</td></tr><tr><td>胎牛血清</td><td>临时机场管理局实验室</td><td> A15 - 351 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Mediatech </td><td> 30 - 002 - CI </td><td></td></tr><tr><td>两性霉素B </td><td> Mediatech </td><td> 30 - 003 - CF </td><td></td></tr><tr><td>孵化器</td><td> Thermo Scientific的</td><td> 3597 </td><td>新机场工程统筹署8000系列WJ W / O2抑制</td></tr><tr><td>胰蛋白酶EDTA </td><td> Mediatech </td><td> 25 - 052 - CI </td><td></td></tr><tr><td>轨道摇床</td><td> VWR </td><td> 12620-926 </td><td></td></tr><tr><td>紫外灯</td><td>三洋Denri </td><td> FLR40SBLB / M </td><td>可容纳两个40W，365nm的黑光灯蓝色紫外线灯泡</td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5811 000.010 * </td><td> *订单号。 型号5810 ř </td></tr><tr><td>显微镜</td><td>尼康</td><td> TI - ND6 - PFS </td><td>随着556nm激发/ 586nm排放filterset </td></tr></table>

tracking hypoxic signaling within encapsulated cell aggregates

在1型糖尿病，胰腺β细胞中胰岛素的生产损失和潜在的致命性高血糖的结果的自身免疫性破坏。作为一种替代治疗选择外源性胰岛素注射，功能胰腺组织移植已探索 1,2 。这种方法提供了更自然，恢复正常血糖长期承诺。从宿主的免疫系统是必需的捐助组织的保护，以防止排斥和封装是用来帮助实现这一目标的方法。 生物衍生材料，如海藻酸钠3和琼脂糖4，已经胶囊建设传统的选择，但可能会引起炎症或纤维化繁茂5可以阻碍养分和氧气的运输。另外，合成聚乙二醇（PEG）凝胶的非降解，易功能化，高纯度，可控孔径，是非常具有生物相容性，6,7,8。作为一个额外的好处，PEG水凝胶可迅速形成一个简单的光交联反应，不需要申请非生理温度6,7。这里描述的是这样一个过程。在交联反应，光引发剂的UV退化，1 - [4 - （2 - 羟乙氧基）苯基] -2 - 羟基-2 - 甲基丙烷- 1 -（Irgacure 2959），产生自由基攻击乙烯的碳 - 碳双dimethacrylated PEG（PEGDM）诱导交联的债券链的末端。在10分钟内就可以实现交联。以这种方式建造的聚乙二醇水凝胶已被证明有利的支持细胞 7,9，和光引发剂浓度低，并简短地暴露在紫外线的照射是不会损害10封装的组织的活力和功能。虽然我们甲基丙烯酸聚乙二醇与方法如下所述，PEGDM也可以DIRectly从Sigma公司，如供应商购买。 一个封装的固有后果是细胞从血管网的隔离。营养物质的供应，特别是氧气，因此，减少和限制扩散。这减少的氧气供应，尤其是可能影响β细胞的胰岛素分泌功能是高度依赖氧气 11-13 。胶囊成分和几何也将影响对氧的扩散速度和长度。因此，我们还描述了确定乏氧细胞内的聚乙二醇胶囊的技术。与重组的腺病毒的细胞感染允许一个荧光信号产生时，细胞内缺氧诱导因子（HIF） 途径激活14。由于HIFs转录缺氧反应的主要监管机构，他们所代表的理想目标标记检测缺氧信号15。这种方法允许hypoxi简便，快速检测C细胞。简单地说，腺病毒有一个缺氧反应元件（HRE）三聚体的控制下，红色荧光蛋白的序列（DS红DR自​​Clontech）。低氧气条件下稳定HIF - 1，将驱动器的荧光蛋白（图1）的转录。对这种病毒的建设更多细节已公布以前 15 。该病毒是存储在10％甘油于-80 ° C的很多150微升1.5 mL离心管分装在一个3.4 × 10 10 PFU / mL的浓度。 在我们的实验室以往的研究表明，MIN6细胞封装为聚合整个文化周在20％的氧气维持其可行性。 MIN6聚集培养2或1％的氧气表明：用溴化乙锭染色，以及相对20％的氧气，细胞形态的变化，坏死细胞的两个标志（仍然可行的约85-90％）。顺利球形显示20％的聚集是洛杉矶T和聚合似乎更喜欢杂乱无章的细胞群体。虽然含氧量低应力不会导致在生存能力明显下降，这显然是影响MIN6聚集和功能为测量血糖刺激胰岛素分泌15。封装的细胞在20％和1％的氧气西方印迹分析还显示，为DsRed的议员蛋白表达与低氧条件下培养的细胞中HIF -1α的显著增加。

Watch this Scientific Journal Video about Tracking Hypoxic Signaling within Encapsulated Cell Aggregates at JoVE.com

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

封装在一个交联的聚乙二醇水凝胶的细胞照片的方法描述。使用荧光标记系统内缺氧的信号封装小鼠胰岛素（MIN6）聚合跟踪。这个系统允许在水凝胶支架和相关的缺氧信号串行检查细胞与细胞表型的变化。

在封装的细胞聚集跟踪缺氧信号

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

tracking-hypoxic-signaling-within-encapsulated-cell-aggregates

Research

JoVE Journal

Bioengineering

10.5K 次观看.  University of South Carolina. 封装在一个交联的聚乙二醇水凝胶的细胞照片的方法描述。使用荧光标记系统内缺氧的信号封装小鼠胰岛素（MIN6）聚合跟踪。这个系统允许在水凝胶支架和相关的缺氧信号串行检查细胞与细胞表型的变化。

视频: Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

科研

生物工程

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 ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力：受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels.
The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

The motion regimes of ibuprofen encapsulated in &#946;-cyclodextrin nanosponges polymer network are investigated using pulsed-field-gradient spin-echo (PGSE) NMR technique. Synthesis, purification, drug loading, implementation of the NMR pulse sequence and data analysis to work out the mean square displacement of the drug at several observation times are described in detail.

布洛芬封装的输运性质环糊精Nanosponge凝胶：质子HR-MAS NMR光谱研究

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

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, ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

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 ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

o 型血管哺乳动物细胞产生均匀集料的轨道晃动培养

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...